Ir
OPTICAL ACTIVITY AND
LIVING MATTER
by
G. F. GAUSE
Professor of Experimental Blologij
UNIVERSITY OF MOSCOW
No. 2 of a series of monographs on general physiology
edited by B. J. Luyet
Published by
BIODYNAMICA, Normandy^ Missouri
1941
"^^Ai
LI8RAR Y| ^
•f-^WCJ^lUK^^)'
Judging by the bibliographical list of publications deal-
ing with optical activity in biological material, one has
the impression that this subject did not arouse among
American scientists the same degree of interest as did
many other problems of biophysics and biochemistry. It
is thought that this review will contribute to focus the
attention of more of the so active investigators of this
country on the important role of asymmetry in the build-
ing stones of protoplasm.
In presenting ''(9p^«c«^ Activity and Living Matter" by
G. F. Gause to scientists at large, the object of the editor
of this series of monographs is to bring to the fore a
subject w^hich seems to be of fundamental significance in
the problem of the structure and the mechanism of action
of living matter.
The contents of this monograph with the exception of
the General Bibliography, are reprinted from BIODY-
NAMICA, Nos. 52 and 56, 1939; 62 and 63, 1940; 70 and
71, 1941.
The Editoe.
Saint Louis, Missouri, May 1941.
••(•-
PREFACE
Althongli the study of the asymmetry of protoplasm
was begun by Louis Pasteur about a century ago, it did
not receive from the biologists the attention which it de-
serves. The observations on that subject are scattered
and need to be brought together into a separate division
of experimental biology. The author of the present mon-
ograph, who has for several years been engaged in expe-
rimental studies of the structure and of the activity of
living systems as related to the asymmetric configuration
of their constituents, intends to review here this scat-
tered literature and to discuss the various problems which
the subject involves.
Pasteur would, no doubt, rejoice in the importance that
a number of questions related to the asymmetry of proto-
plasm have acquired in the development of medical
sciences. The recent findings on anthrax, a subject to
which Pasteur has contributed so much, illustrate this
point. Bruckner and Ivanovics showed in 1937, in the lab-
oratory of Professor Szent-Gyorgyi, that the unnatural
optical isomer of glutamic acid, which was not found
anywhere before in organic nature, enters into the compo-
sition of the capsules which enclose the anthrax bacilli.
The capsules are responsible for the virulence of the
bacilli, and the investigators just mentioned suggest that
the protective role of the capsules is due to the unnatural
configuration of glutamic acid.
The author wishes to express his thanks to Professor
W. W. Alpatov (Moscow) and to Professor W. J. Ver-
nadsky (Moscow) for their aid in the course of the prep-
aration of this work, and to Professor B. J. Luyet (St.
Louis) for the revision of the manuscript.
G. F. Gause.
TABLE OF CONTENTS
Page
PREFArE - 4
TABLE OF CONTENTS 5
PRELIMINARY CHAPTER
PRINCIPLES AND DEFINITIONS
1. Dissymmetry and Asymmetry 9
2. Optical and Geometrical Asymmetry 11
3. Dissymmetric Structure as a Basis of Optical Ac-
tivity - - 12
4. "Eelative Configuration" and "Biological Series"
of Optical Isomers - 15
CHAPTER I
OPTICAL ACTIVITY OF BIOLOGICAL MATERIAL
1. Dissymmetry in Organic and in Inorganic Nature.— 19
2. Asymmetry as a Specific Property of Protoplasm.— 20
3. Asymmetry of Primary Constituents of Protoplasm 21
4. Asymmetry of Secondary Constituents of Proto-
plasm 24
5. Exclusiveness of Asymmetry-Sign in Primary Sub-
stances 27
6. Non-Exclusiveness of the Asymmetry-Sign in Sec-
ondary Substances 28
7. Relative Configuration of Biological Material 30
8. Asymmetry as a Criterion of the Organic Origin
of a Substance 31
CHAPTER II
THE ORIGIN AND MAINTENANCE OF OPTICAL
ACTIVITY IN LIVING MATTER
1. The Transmission of the Asymmetric State by
Asymmetric Synthesis 35
2. The Transmission of Asymmetry, from the Ther-
modynamic and Kinetic Point of View 37
6
3. Maintenance of Optical Purity by the So-Called
''Stereo-autonomic Substances" 43
4. Procedures Used by Nature for Maintaining Opti-
cal Puritv and Establishing a ''Fixed Internal
Milieu" ■ 45
5. Biological Advantages of Optical Purity 51
6. The Origin of the Asymmetry of Protoplasm. 52
7. General Survey of the Problem of the Origin and
Maintenance of Optical Asymmetry 53
CHAPTER III
HEREDITY AND THE INFLUENCE OF ENVIRONMENTAL
FACTORS ON THE OPTICAL ACTIVITY OF
BIOLOGICAL MATERIAL
1. The Impossibility of Inverting the Optical Prop-
erties of the Primary Constituents of Proto-
plasm 59
2. The Impossibility of Modifying Protoplasm so as
to Cause it to Invert the Optical Properties of
the Products of its Metabolism 61
3. Mechanisms Controlling the Production of a Given
Optical Isomer 67
a. Production of Dissyinmetrir Substances from
Symmetric Phenyl-Glyoxal 67
&. Production of Optical Isomers by Esterases 68
c. Production of Optical Isomers bv Oi)tically Active
Alkaloid Catalysts ' *.... 68
d. Production of a Given Optical Isomer by a Chemi-
cal Alteration of the Catalyst .'. 69
e. Control of the Production of Optical Isomers by
Intermediate "Pathways'' 70
/. Control of the Production of Optical Isomers by
an Inversion of the Waldeu Tyije 73
CHAPTER IV
ON THE RELATION BETWEEN THE INVERSION OF
SPIRALLY TWISTED ORGANISMS AND THE
MOLECULAR INVERSION OF THEIR
PROTOPLASMIC CONSTITUENTS
1. Morphological Dissymmetry and Morphological In-
version - 79
2. Mori)li()l()ij;ical J)issyiinnetry and Morphological In-
version mBacillus Mycoides 81
3. Morphological Dissymmetry and IMorphological In-
version in the Snail, F ruticicola lantzi 83
4. Some Physiological Properties of the Dextral and
the Sinistral Strains of Bacillus mycoides 84
5. Some Physiological Properties of the Dextral and
of the Sinistral Strains of the Snail, Fruticicola
lantzi 89
6. On the Relation between Morphological Inversion
and Molecular Inversion..... 91
7. Morphological Inversions and the Theory of Spiral
Growth _ i 93
CHAPTER V
ANALYSIS OF VARIOUS BIOLOGICAL PROCESSES BY
THE STUDY OF THE DIFFERENTIAL
ACTION OF OPTICAL ISOMERS
Asymmetric Analysis 99
Section- I. Analysis of the Mechanism of Toxic
Action,
1. Toxic Action of the Optical Isomers of Nicotine 100
2. Toxic Action of the Optical Isomers of Organic
Acids - 108
Section II. Analysis of the Evolution of the
Nervous S^^stem. I
1. Stereo-coefficients of Action of the Optical Isomers
of Nicotine in the Phylogenetic Series 116
2. The Acetylcholine System and the Differential Ef-
fect of the Optical Isomers of Nicotine 120
Section III. Analysis of the Mechanism of Va-
rious Physiological Functions in Protozoa.
APPENDIX
ASYMMETRY OF PROTOPLASM AND THE
STRUCTURE OF THE CANCER CELL \
GENERAL BIBLIOGRAPHY 133
SUBJECT INDEX 154 /^^^
AUTHOR INDEX 157 - * ■
1 2:
PRELIMINARY CHAPTER
PRINCIPLES AND DEFINITIONS
1. Dissymmetry and Asymmetry. A survey of the litera-
ture on optical activity of protoplasm reveals some confu-
sion in the terminology. Terms such as dissymmetry and
asymmetry, which are so often used, are not always clearly
defined. Some preliminary definitions are, therefore,
necessary.
Dissymmetry is a property of the individual components
of a system, that is, in the cases to be considered here, a
property of molecules, while asymmetry refers to an aggre-
gate of molecules.
The term dissymmetry was used in this sense for the first
time by Pasteur in the classical paper that he wrote in 1848
on the relations between crystalline form, chemical compo-
sition and optical rotation and that he summarized in the
two well-known lectures delivered in 1860 before the Paris
Chemical Society on the molecular dissymmetry of natural
organic products. Pasteur says that when we study mate-
rial objects of whatever nature, as regards their form and
the repetition of their identical constituent parts, we soon
recognize that they fall into two large classes which present
the following characters. Those of the one class, placed
before a mirror, give images which are superposable on
the objects themselves, while the images of the others are
not superposable on the objects. A cube, straight stairs, a
branch with opposite leaves, the human body — these are of
the former class ; an irregular tetrahedron, winding stairs,
a hand — these belong to the second group. The latter are
10 PRINCIPLES AND DEFINITIONS
dissymmetric,^ and are defined as objects possessing non-
superposable mirror images.
Dissymmetric objects can exist in two forms, right and
left.
When the two forms of dissymmetric molecules are rep-
resented in equal concentrations (racemic mixture),' the
aggregate of molecules is symmetric. When they are rep-
resented in unequal concentrations, the aggregate is non-
symmetric. There may be a predominance of the right
forms (dextrality) or of the left forms of molecules (sinis-
trality). Pasteur did not propose any special term for such
a deviation of the molecular aggregate from the racemic
state. His views on this subject were somewhat uncer-
tain.' Following Emil Fischer (1894) and Japp* (1898),
we shall designate this condition by the term asymmetry.
1 In the German (1891) and in the English (1897) translations of Pasteur's
work (lectures of 1860), the word dissymmetry was arbitrarily replaced by
the word asymmetry.
2 Eecently Findlay (1937) pointed out that the term acide racemique, as
applied to tartaric acid, was due to Gay-Lussac (1828), but its use in the
sense accepted at present originated with Pasteur (1861). Pasteur wrote in
1860 : ' ' We still need a word in chemical terminology to express the fact
of a double molecular dissymmetry concealed by the neutralisation of two
opposite dissymmetries, the physical and geometrical effects of which com-
pensate each other exactly. ' '
3 Pasteur did not distinguish sharply the dissymmetry of individual mole-
cules from the asymmetry of their aggregates in the sense given above. For
him the molecules acquire dissymmetry by receiving a ' ' twist ' ' in living
organisms or in contact with products of living organisms and they lose their
dissymmetry by being untwisted. In 1860 he wrote that "the twisted organic
group can be untwisted and so assume the ordinary character of artificial and
mineral substances. ' ' The ' ' twisting ' ' was considered as characteristically
' ' vital ' ' and destructible by energetic chemical reactions. According to mod-
ern views, these reactions, instead of ' ' untwisting ' ' the molecules, produce a
racemisation or an equalization in the concentrations of the right and left
forms of a substance. While Pasteur is the discoverer of the fact that
' ' Racemic tartaric acid of chemists, inactive as to the optical rotatory power,
consists of two acids, the rotations of which mutually neutralize each other,
as one of them rotates to the right and the other to the left, and both in the
same degree," (Pasteur, 1848, p. 458) he thought that the molecules of the
racemates were symmetric by their very nature, and that they became dis-
symmetric in their separation from the racemate by crystallization of the anti-
podes under the action of some dissymmetry forces which might be furnished
by ' ' organic dissymmetric particles on the surface of the crystallization dish ' '
PRINCIPLES AND DEFINITIONS 11
From what has been said, it follows that dissymmetry,
or noii-siiperposability of mirror image on the original
object, can exist without any asymmetry, as in racemic
mixtures. Dissymmetric molecules have the possibility
of forming symmetric or asymmetric aggregates ; asym-
metry is the realization of one of these two potentialities.
It is, therefore, obvious that dissymmetry represents a
necessary pre-requisite condition for any asymmetric state.
2. Optical and Geometrical Asymmetry. Asymmetry as
defined here should be distinguished from geometrical
asymmetry. A geometrically asymmetric figure is one
which possesses no element of symmetry, that is, no center,
no axis and no plane of symmetry, while dissymmetric
figures (in the sense of Pasteur) might possess a complex
system of axes of symmetry, although they cannot possess
(Pasteur, 1884). According to our views, one half of a racemic aggregate
consists of the right and the other of the left form of molecules, before, as
well as after, crystallization.
That Pasteur was mistaken in this particular point is evidenced by the fol-
lowing investigations. Ostwald (1889) has shown by electric conductivity
methods that, in dilute water solutions, racemic tartaric acid does not exist
as such but is entirely dissociated into its dextrorotatory and laevorotatory
components. Eaoult reached the same conclusion by cryoscopic methods.
Wyrouboff (1884), Jungfleisch (1884), and Errera (1898) pointed out that
the separate crystallization of antipodes from racemic tartrate depends on the
relative solubilities of the individual components and of the mixture. These
solubilities, in their turn, are controlled by the temperature. Thus, at ordi-
nary room temperature the antipodes are less soluble than the racemic mix-
ture, and they crystallize separately, while, at temperatures above 26° C, the
order of solubility is reversed, and the racemate crystallizes.
4 Emil Fischer (1894) introduced the concept of asymmetric synthesis, that
is, of the production of molecules which exhibit a rotation of a given sign
with full or partial exclusion of the antipode. But the term asymmetry for
expressing the properties of aggregates of molecules was employed^ — for the
first time, it seems — by Japp (1898) in his well known address, "Stereo-
chemistry and Vitalism," which was followed by an interesting discussion in
' ' Nature. ' ' Japp wrote that the simultaneous production of two opposite
asymmetric halves is equivalent to the production of a symmetric whole,
whether the two asymmetric halves be actually united in the same molecule,
as in the case of meso-tartaric acid, or whether they exist as separate mole-
cules in the loft and right constituents of racemic acid. This statement shows
quite clearly that the author conceived asymmetry as the property of the
aggregate of molecules and not as the configurational character of the indi-
vidual molecules (the term enantiomorph was used in this latter sense).
12 PRINCIPLES AND DEFINITIONS
a plane, a center or an alternating axis of symmetry {cf.
the definition of Lowry, 1935), these elements being incom-
patible with the non-superposability of the image. So,
dissymmetric molecules are not necessarily asymmetric in
the geometrical sense.
3. Dissymmetric Structure as a Basis of Optical Activ-
ity. The fact that the rotation of the plane of polarized
light is caused by a dissymmetric structure of molecules
leaves no place for doubt, but the problem of the physical
mechanism by which this is done did not yet receive a defi-
nite solution. Two models proposed by Pasteur — irregu-
lar tetrahedron and spiral line — have formed the basis for
further theories. We shall consider separately the case in
which optical activity is due to a dissymmetric spatial dis-
tribution of atoms as found in entire crystals and the case
in which it is due to a dissymmetric structure of isolated
molecules.
It is known that the optical activity of quartz depends
on the structure of the crystal itself, since the rotation of
the plane of polarized light disappears with the crystalline
state. The optical effect also diminishes, and at last van-
ishes when a plate cut out from a crystal of quartz passes
from a position perpendicular to the direction of the ray
to an inclined position. Consequently, the fundamental
difference between the dissymmetry of quartz and the mo-
lecular dissymmetry of organic substances lies in the fact
that in the former case the crystal as a whole is anisotropic,
i.e., possesses different properties in diiferent directions,
while, in the latter, as it was ascertained by Pasteur, dis-
symmetry represents a property of the separate molecules
independent of their relative position in space. A sub-
stance in which one of the two possible dissymmetric forms
of molecules, right or left, predominates, will possess
optical activity.
It was Fresnel (1824) who suggested for the first time,
that the structural dissymmetry of quartz may be ex-
plained on the basis of the spiral distribution in space of
the molecules of silicon. In one of the two optical anti-
PRINCIPLES AND DEFINiriONS 13
podes of quartz, these spirals would turn from right to
left and, in the other, from left to right. This view was
adopted by Pasteur (1860), and, about a hundred years
after its formulation by Fresnel, it received full confirma-
tion in the X-ray analysis of quartz made by Bragg (1913,
1925). This investigator showed that crystals of quartz
can be considered as giant molecules in which the constitu-
ent units build up a three-dimensional network, w^liere
every atom of silicon is linked to four atoms of oxygen,
wiiilst every atom of oxygen unites two atoms of silicon.
The complex aggregate thus formed has a spiral structure
which is shown in Fig. 1. The lines uniting the centers of
Fig. 1. Spiral structure in a crystal of quartz. The silicon atoms are
represented by solid black circles, the oxygen atoms by lighter and larger
circles. Three atoms of silicon form a spire. Each atom of silicon is in
the center of a tetrahedron at the apices of wliicli are 4 oxygen atoms; only
2 of the latter are represented in the figure.
the atoms are spirals, and these spirals are twisted in
opposite directions in dextrorotatory and in laevorotatory
quartz. (For further details on the coordination of sepa-
rate spirals in the so-called a and 3 form of quartz, cf.
Bragg.) Let it be noticed, then, that it is the spiral type
of structure which prevails in the dissymmetric spatial
distribution of elements in crystals of quartz.
What is the structure of dissymmetric organic molecules
and its relation to optical rotation? Modern theories, a
detailed account of which may be found in the excellent
monograph by Lowry (1935), consider the irregular tetra-
14
PRINCIPLES AND DEFINITIONS
lieclron with four different radicals situated in its corners
as the basis for the explanation of the origin of optical
activity. This structure accounts for both the existence
as well as the approximate value of optical rotation in the
simplest dissymmetric molecules. It should be noticed
that a tetrahedric molecule presents a spiral type of dis-
tribution of its atoms. In Fig, 2 (a) is represented an
Fig. 2. Dissymmetric configuration of organic molecules; a) 1-isomer,
b) d-isomer.
irregular tetrahedron in the corners of which are placed
four different groups. In the order of diminishing mag-
nitude these groups can be arranged in the following-
manner: Ri > R2 > R3 > R4. By joining the centers of
these groups in the order just given a spiral is obtained.
If the largest group (Ri) is placed nearest to a hypotheti-
cal observer, the spiral represented in Fig. 2 (a) will
appear to rotate counter-clockwise. According to Boys
(1934), such a structure would correspond to the left abso-
lute configuration of the molecule. If we interchange the
groups R2 and Rs, we obtain a figure which is the mirror
image of the preceding one ; the spiral twist will now as-
sume a clockwise direction and the molecule will possess
the right configuration.
Recently an attempt has been made to adapt the concept
of absolute configuration to the definition of the configura-
PRINCIPLES AND DEFINITIONS 15
tioii of natural a-amiiio-acids (see Eainey, 1937). We
shall also mention as related to this problem the geometri-
cal investigations of Study (1913) on the right and left
structures in a system of points. Finally we wish to point
out again that the spiral distribution of elements appears
as basic in the mechanism of optical rotation in molecules
as well as in crystals.
4. ^'Relative Configuration" and ''Biological Series"
of Optical Isomers. Emil Fischer (1894) drew attention
to the necessity of distinguishing the relative configura-
tion of a substance from the sign of its optical rotation,
there being substances which possess the same relative
configuration but rotate the plane of polarized light in
opposite directions. The importance of this remark be-
came more evident in the subsequent developments of
stereochemistry. Changes in temperature, solvent, con-
centration, etc., are often accompanied by a change in the
sign of the optical rotation. As Lowry (1935) pointed out,
these changes make it impossible to judge the configura-
tion of a substance by the sign of its rotation. This may
be demonstrated by the following example. Let us con-
sider an optically active compound
CH3 X
\ /
c
/ \
/ \
containing a single asymmetric carbon atom, linked to
methyl and ethyl and to two other radicals, X and Y. No
matter what the influence of temperature and of solvent
is, the sign of the rotation will be reversed but its magni-
tude will be unaltered if the methyl and ethyl radicals are
interchanged, i.e., if usual optical inversion takes place.
The rotation will disappear completely if methyl is re-
placed by a second ethyl radical, or conversely, since then
the plane of symmetry will appear in the molecule. If
methyl is replaced not by ethyl but by propyl, it is gen-
16 PRINCIPLES AND DEFINITIONS
erally admitted (Lowry, 1935) that the sign of the rotation
will be reversed, i.e., that the molecules
CH, X C,H, X
\ / \ /
\ / \ /
C and C
/ \ / \
/ \ / \
C,H, Y C,H, Y
will have opposite rotations, although the position of the
univalent radical C2H5CXY is identical, and although there
has been only a substitution of one chemical group in the
molecule by another. Such possibilities render illusory
any conclusion as to the configuration of a substance on
the basis of the direction of its rotation.
To clarify this situation, Fischer (1894) proposed to
take as a prototype of configuration that of a specific
isomer of some definite substance and compare to this pro-
totype the optical isomers of other substances. In this
manner a series of optical isomers of different substances
can be established, all the members of this series possess-
ing the same relative configuration. Wohl and Freuden-
berg (1923) suggested that the members of one such series
be designated by the letter d and their antipodes by the
letter /, while the sign of their optical rotation w^ould be
indicated by (+) for a rotation to the right and by (-) for
a rotation to the left. The decision as to which one of the
two series should be marked by the letter d is, of course,
arbitrary, the absolute configuration of the substance being-
unknown. According to this system, a substance belong-
ing, for example, to the left steric series, but rotating the
plane of polarized light to the right will be marked by I (+).
Fischer, furthermore, suggested to take as a standard of
comparison dextrorotatory glucose, conventionally taking
it as a r/-form. He proposed that, in writing the formulas,
the aldehydic or ketonic group of sugars and the carbonyl
group of monobasic acids be put on top and the chain of
carbon atoms in a downward direction, the hydroxyl of the
fifth carbon atom being to the right. If one figures out.
PRINCIPLES AM) DEFINITIONS 17
on llie basis of what is known on chemical structure, wliieh
isomer of fructose presents the same position for the tifth
carbon atom as rf-glucose, one finds that it is laevorotatory
fructose. Thus d (+) glucose and d (-) fructose possess
the same relative configuration in spite of their rotation
in opposite directions. Both these isomers are found in
living organisms and belong to the same *' biological
series."
Wohl and Freudenberg (1923) proposed to take glycer-
ine aldehyde and not glucose, as a standard of comparison,
conventionally considering the dextrorotatory form as a
member of the <7-series and attributing to it such a struc-
ture that the hydroxyl of the fifth carbon atom be again
written to the right.
SUMMARY
1. Dissymmetry is the property of molecules of possess-
ing non-superposable mirror-images. Dissymmetric mole-
cules can exist in two forms, right and left. 2. Asymmetry
is the property of molecular aggregates of presenting a
predominance of the right or the left form of dissymmetric
molecules. 3, Optical asymmetry is to be distinguished
from geometrical asymmetry. 4. Optical activity is at-
tributed to the spiral arrangement of atoms, either in
entire crystals, as in quartz, or in single molecules, as in
some organic compounds. 5. If, besides the sign of the
optical rotation of a substance, one considers the config-
uration of its molecules, one can classify the optical isomers
into "biological series" as found in living organisms.
BIBLIOGRAPHY
BEAGG, W., Proc. Boi/. Soc. A., 89, 575, 1913; IM, 405, 1925.
EERERA, G., Nature, 58, 616, 1898.
FINDLAY, A., Nature, 140, 22, 1937.
FISCHER, E., Ber. cliem. Ges., ^7, 3189, 1894; 3^, 3638, 1900.
FRESNEL, A., Bull. Soc. Philomat., p. 147, 1824.
GAY-LIJSSAC, L., Couvs de Chimie, Paris, 1828.
JAPP, F. R., Nature, 58, 452, 1898.
JUNGFLEISCH, M. E., Bull. Soc. Chim. Paris, 41, 222, 1884.
LOWEY, T. M., Optical Eotatoiy Power, Longmans, Green & Co., Loudon,
1935.
18 PRINCIPLES AND DEFINITIONS
OSTWALD, W., Z. physikal. Chem., 3, 369, 1889.
PASTEUR, L., Ann. Chim. et Phys., 24, 442, 1848; 61, 484, 1861.
, Recliei'clies sur la Dissymetrie Moleculaire des Produits Or-
ganiques Naturels. Soc. Chim. Paris. English translation in Alembie
Club Beprints, 14, 1860.
, Bev. Scieniif. Hi, 4, 2, 1884.
RAINEY, R. C, Nature, 140, 150, 1937.
STUDY, E., Arcli. Math, unci Physilc, 31, 193, 1913.
WOHL, A. AND FREUDENBEEG, K., Ber. chem. Ges., 56, 309, 1923.
WYROUBOFF, G., Bull. Soc. Chim. Paris, 41, 212, 1884.
CHAPTER I
OPTICAL ACTIVITY OF BIOLOGICAL MATERIAL
1. Dissymmetry in Organic and in Inorganic Nature. It
has been repeatedly pointed out that all physiologically
important substances possess a dissymmetric structure.
This is precisely what Pasteur meant when he wrote: "On
trouve la dissymetrie etablie notamment dans les principes
immediats essentiels a la vie. ' ' But is there any essential
relation between dissymmetry and life, in the sense that
one is a necessary attribute of the other! Dissymmetry
is certainlj^ much more general than life. We know that
the dissymmetric structure exists in crystals of quartz.
The same is true of several metallic compounds {cf. Lowry,
1935). Recently Jaeger (1919), after having investigated
a great number of inorganic compounds, came to the con-
clusion that the dissymmetric structure might be much
more general than we usually assume, but that, in inorganic
nature, the existence of dissymmetry is often difficult to
establish, there being no method for separating the anti-
podes. Vernadsky (1934) made a similar remark. In
such cases at least, dissymmetry^ has no obvious relation
to life. But, if dissymmetry exists without life, life might
not exist without dissymmetry. The possibility that life
be the attribute of systems built of substances of such a
level of complexity that dissymmetry is the very condition
of their existence is not excluded. A suggestion which was
recently made by Ackermann (1935), and which is practi-
cally identical with that of Pasteur (1884), is that dissym-
metry is characteristic of the basic components of proto-
plasm, whilst such products of metabolism as urea, uric
acid, creatinin and hippuric acid are devoid of dissym-
metry and their molecules are structurally inactive. The
simplest amino-acid of the protein molecule, glycocoll, is
[on-metals
C N
Si P
S
As
Se
Te
20 OPTICAL ACTIVITY OF BIOL. MATERIAL
the only one devoid of dissymmetry, and, in metabolic
processes, it is less important than the other amino-acids
which are dissymmetric.
It is of interest to mention here that the elements of
which optically active compounds consist include twenty-
one of them, as follows (Lowry, 1935) :
Metals
B C N Be
Al
Cr Fe Co Ni Cu Zn
Ell Eh
Ir Pt
2. Asymmetry as a Specific Property of Protoplasm. It
is generally established that all the substances which are
produced in the laboratory or in nature, without the action
of living organisms, have right and left forms represented
in equal concentrations, the formation of both being equally
probable. It has never been observed, for example, that
in any quartz bed the right or the left crystals would pre-
dominate to any extent (Tromsdorff, 1937; Lemmlein,
1938). There is, of course, dissymmetry in individual
components, but no asymmetry in their aggregation.
On the other hand, all basic chemical substances of which
living systems are made up or which are formed in connec-
tion with the activity of living systems, deviate from the
racemic state and are represented mainly by one antipode.
In other words, asymmetry is a specific attribute of living-
systems and an essential feature of their organization.
This is one of the most significant principles of experi-
mental biology ; it is based on a large number of observa-
tions accumulated within the last hundred years, since the
pioneer work of Pasteur.
We shall study here, in some detail, which parts of living-
systems consist of racemic compounds and which parts
deviate from the racemic state and in what direction. It
will appear that the asymmetric state of protoplasmic
components is directly related to the role played by these
components in metabolic activity.
OPTICAL ACTIVITY OF BIOL. MATERIAL 21
3. Asymmeiry of Primary Constituents of Protoplasm.
From the view-point of their asymmetric molecular aggre-
gation, the substances which enter into the composition of
living systems may be divided into two groups. Physio-
logists have for a long time been accustomed to call these
two groups, respectively, the primary and the secondary
constituents of protoplasm. To the group of primary sub-
stances belong the proteins and the lipoids which form
together the so-called lipoprotein complexes, and the carbo-
hydrates which functionally are closely related to them.
These primary substances, except for some stored carbo-
hydrates and proteins, build up protoplasm itself and pre-
side over the fundamental living processes. To the group
of secondary constituents belong various products of trans-
formation of the primary substances, which represent
either storage material or excreta.
We shall study, to begin with, the asymmetric structure
of primary substances, and we shall consider, first, the
degree in which they deviate from the racemic state, or, in
other words, their optical purity.
As far as proteins are concerned, the optical activity of
which was already known to Pasteur, Emil Fischer was the
first to express the idea that their constituent amino-acids
are always found in protoplasm in the optically pure state,
and that, when a total or partial racemisation occurs, it is
due to the application of too coarse methods of isolation.
For instance, serine from silk was known for a long time
only in the form of a racemic compound, as it is rather
easily racemised in the process of protein hydrolysis.
However, Fischer (1907) succeeded in isolating from silk
optically active serine, of which the specific rotation (in
hydrochloride solution,^ at 18°) was + 11.6°, while the rota-
tion of optically pure laevorotatory serine prepared syn-
thetically {i.e., crystallized from racemate with alkaloids)
was + 14.4° (at 20°). On the ground of these experimental
data alone it is, of course, impossible to conclude that serine
in silk is optically pure, and that it is partially racemised
1 The hvdroc'liloride of laevorotatory serine is dextrorotatory.
22 OPTICAL ACTIVITY OF BIOL. MATERIAL
ill the process of isolation, but we shall see below that the
principle itself is definitely established.
Pringsheim (1910) had shown on asparagine that the
optically pure form is gradually racemised by boiling with
water, a step in the isolation procedure.
With appropriate treatment, amino-acids both of vege-
table and animal origin, always prove to be optically pure,
that is, one isomer only of each amino-acid is present, its
antipode being completely absent. In the case of leucine,
this was established by the elaborate investigations of
Ehrlich and Wendel (1908), the results of which are given
in Table 1.
TABLE 1
Specific Eotation of Preparations of Leucine of Different Origin,
IN Water at 20° (Ehrlich and Wendel, 1908)
Synthetic, optically pure preparation - 10.3°
From egg-wliite (chicken) - 10.4°
From casein (cow's milk) -10.3°
From yeast {Saccharomyces cerevisiae) - 10.8°
The optical purity of tyrosine remained for a long time
questionable as a result of a number of old contradictory
observations (Lippman, 1884). But Schulze and Winter-
stein (1905) have definitely shown that, after careful
preparation from vegetable material, one obtains always
optically pure substances and that racemisation and the
consequent decrease of rotatory power are the result of
the application of coarse methods of isolation (Table 2).
TABLE 2
Optical Eotation of Preparations of Tyrosine of Different
Origin, in Hydrochloride Solution
Synthetic, optically pure prep-
" aration - 16.4° Fischer, 1900
From Cow 's Milk ; hydrolysis | - 13.2°
of casein by HCl \ - 11.6° ' '
From the bulbs of Dahlia
variabilis; 80% boiling
alcohol was used in the
isolation procedure
- 12.5° Schulze and Winterstein, 1905
- 12.9°
From embryos of Liipinus
albvs; autolysis - 16.2^^
OPTICAL ArTTYTTY OF BIOL. MATERIAL 23
The pveparation from cow's milk and that from the
bulbs of Dahlia show a weak racemisatioii as a result of
the treatment, while the preparation from the embryos of
Lupinus is optically pure ; the autolysis procedure used in
this last case prevents racemisation.
One can, at present, consider as an established fact that
all amino-acids entering into the composition of proto-
plasmic proteins are optically pure ; not a single exception
is known/
The fats or lecithins, which contain nitrogen and phos-
phorus, and which are considered integral constituents of
the fundamental units of protoplasm, are also optically
pure, as it was, for instance, established by the investiga-
tions of Mayer (1906).
Among primary substances, the carbohydrates, as well,
are for the most part optically pure. Brown and Morris
(1893) have shown in an extensive investigation that glu-
cose and other sugars are found in the optically pure form
in the leaves of the plant Tropaeolum majus.
An interesting exception to the general rule has been
observed in sugars. Neuberg (1900) found in the human
organism optically inactive, racemic sugar under patho-
logical conditions. Salkowsky (1892), who had discovered
that in this case a pentose (arabinose) is excreted in urine,
instead of glucose as it happens in glucosuria, called the
disease pentosuria. Neuberg established that the arabi-
nose excreted in urine is optically inactive. These obser-
vations were later confirmed by a number of other physi-
ologists. In what relation the inactive arabinose stands
to the active arabinose entering into the composition of the
nucleo-proteids of our body is at present unknown.
Racemic sugar, dl-galactose, was also found in plants.
Oshima and Tollens (1901) isolated it from the Japanese
marine alga, Porphyra laciniafa.
The presence of racemic sugars in plants and animals is
1 It seems preferable, for the present, to suspend judgment on the recent
data of Kogl and Erxleben (1939) concerning partial racemisation of some
amino-acids in proteins of malignant cells.
24 OPTICAL ACTIVITY OF BIOL. MATERIAL
very rare but it is particularly significant. Since sugars
do not racemise when boiled in water, it seems that the
racemic state does not result from the process of isolation
but that the optically inactive forms actually enter into the
composition of living systems. The origin of racemic
sugars in living organisms is by no means clear. Neuberg
(see Fiirber, Nord and Neuberg, 1920) remarks that it
might not be a mere accidental fact that the two racemic
sugars found are just arabinose and galactose.
To conclude, among the primary substances, all the
amino-acicls, the lecithins and the majority of important
sugars such as glucose, fructose and many others are
always present in protoplasm in the optically pure state.
4. Asymmetry of Secondary Constituents of Proto-
plasm. As one passes from primary to secondary sub-
stances, the optical purity loses its obligatory character.
This is particularly evident in organic acids which repre-
sent intermediate products of metabolism. Their origin
and their signification is still a source of controversy,
especially in plants. Whether, in the latter, the forma-
tion of organic acids is related to the metabolism of the
amino-acids, or whether they represent a stage in the
carbohydrate cycle cannot be decided. When the organic
acids begin to appear, they are optically pure, as if bear-
ing some birth marks from the primary substances, but as
soon as they separate from the primary asymmetric sys-
tem, beginning perhaps to play the role of storage material,
they assume the character of racemic compounds.
The experimental data on which these conclusions are
based are principally those of Ruhland and of his school.^
Ruhland and Wetzel (1929), and later Schwarze (1932)
observed that, in the leaves of different plants, malic acid
is found especially in the two forms: laevorotatory and
racemic (Table 3).
1 The data of the Leipzig school and, particularly, the analytical part of
the work were severely criticized by Bennet-Clark (1937). But, as far as
optical activity is concerned, Euhland's data are reliable. Enzymatic race-
misation of malic acid in plants, according to Bennet-Clark, was observed also
by Naylor (unpublished Thesis, Manchester University, 1935).
OPTICAL ACTIVITY OF BIOL. MATERIAL 25
TABLE 3
Content of Optically Active and of Racemic Malic Acid, in ml. of
Molar Acid Solution per gr. of Dry Weight, in Leaves
OF Different Plants (Schwarze, 1932)
1-malie
dl-malic
acid
acid
0.422
0.163
0.133
0.150
0.869
0.485
0.067
0.208
Nicotiana tabacuvi ..
Pelargonium zonule
P. peltatiini
Pub IIS iclaeiis
According to Rulilancl and Wetzel, the newly formed
malic acid is always optically active and only later does it
pass into the racemic form. In Rheum liyhridum, laevo-
rotatory acid was found to be racemised after the newly
formed portions of it had penetrated into the roots.
Bendrat (1929) observed that all malic acid, in the plant
Sempervivum glaucum, is in the racemic form in the even-
ing, that it increases during the night, and that, after this
increase one can find some laevorotatory acid, in the morn-
ing (Table 4). It seems, then, that the optically active
form appears in metabolic processes and that it is race-
mised later. ■
TABLE 4
Content of Total and Laevorotatory Malic Acid, in ml. of Molar
Acid Solution per gr. of Dry Weight, in the Middle
Leaves of Sempervivum glaucum (Bendrat, 1929)
Total malic l-malic
acid acid
Evening 0.140 0
Morning 0.194 0.013
Data on other organic acids, though incomplete, agree
in general with the observations just mentioned. Thus it
was known to Pasteur that d-tartaric acid as well as dl-
tartaric acid are present in grape juice (see Thiele, 1911).
Inactive lactic acid has been found in the leaves of the
common ash, Fraximis excelsior (Gintl, 1869) and in a
number of other plants (Stoklasa, 1907).
26 OPTICAL ACTIVITY OF BIOL. MATERIAL
Katagiri and Katahara (1937) have shown that, in bac-
teria, optically pure lactic acid is formed first and that it
racemises later under the influence of some environmental
conditions.
Inactive lactic acid was also recorded in comparatively
rare post mortem observations in animals (Morishima,
1900).
As is well known, dextrorotatory lactic acid is found in
vertebrates and in different organs of invertebrates and
racemisation is rare. The tendency has been, for a long-
time, to explain the presence of this racemic lactic acid
(especially in the case of bacterial fermentation) by the
inactivity of the intermediate product, methylglyoxal,
which has no asymmetric carbon atom and from which
racemic lactic acid could be formed without the participa-
tion of an optically active enzyme. But, at present,
methylglyoxal is no longer considered an intermediate
product in the transformation of the carbohydrates, and,
besides, the thorough investigations of Katagiri and Kata-
hara (1937) have demonstrated an initial formation of
active lactic acid, which racemises later.
Racemisation of the secondary substances after they are
detached from the primary asymmetric complex takes place
also in the glucosides which, in plants, play the part of
storage material. The nitrite of mandelic acid which is
enzymatically synthesised in plants in the relatively pure
dextrorotatory form is subsequently racemisecl, and in the
leaves of Primus laurocerasus, a glucoside of racemic dl-
nitrile is found (Kuhn, 1936; this subject will be examined
in detail elsewhere).
The terpenes which, in general, represent vegetable
secretions but on whose origin and physiological function
much remains to be investigated are also often found in
plants in the racemic state. For instance, racemic limo-
nene or dipentene has been observed in Pinus silvestris,
Lauriis camphora, Valeriana ojjic'malis and many others
(Bartelt, 1910, names 16 of them). But optically active
limonene as well is found in the same or similar kinds of
OPTICAL ACTIVITY OF BIOL. MATERIAL 27
plants ; consequently, the secondary origin of the racemic
form from initially active limonene appears to be probable.
The same conld be said also of racemic borneol.
The last group of secondary substances to be considered
is that of the alkaloids. They seem to represent some
moditied fragments of protein molecules which perhaps are
some end products of metabolism. The question of the
optical purity of the alkaloids in plants has been repeat-
edly and extensively discussed. Apparently in a great
number of cases racemisation results from the process of
isolation. This seems to hold, in particular, for optically
inactive atropine, which represents the product of race-
misation of the laevorotatory hyoscyamine, the latter being-
found in plants in the optically active state (McKenzie
and Wood, 1919; Hess and Weltzien, 1920). It is known
that hyoscyamine is very easily racemised by w^eak alkalis
at room temperature. Some alkaloids, however, it was
suggested, might be present in plants in the racemic state,
for instance, coniine and scopoline. Since the racemisa-
tion of these alkaloids proceeds very slowly even at high
temperatures and pressures, an artificial racemisation in
the process of isolation seems excluded (Hess and Welt-
zien, 1920). The origin, in the plant, of racemic coniine
and scopoline is therefore still a mystery.
In spite of the presence of a number of racemic forms
of alkaloids in plants, the majority of them are found in
the optically pure state, for instance, nicotine, anabasine,
etc. The alkaloids constitute, therefore, an exception
among the secondary substances which have severed their
connection with the primary complex. It is probable that,
owing to peculiarities of chemical structure, the mobility
of some groups in the molecule of several alkaloids is ex-
ceptionally low; their optical purity would be due, then,
to a too slow racemisation. In fact, it has not been pos-
sible to attain racemisation of the alkaloid heliotridane by
any of the means employed successfully in other cases
(Menshikov, 1937).
5. Exclusiveness of the Asymmetry-Sign in Primary
28 OPTICAL ACTIVITY OF BIOL. MATERIAL
Substances. As has been said, the primary organic sub-
stances are obligatorily asymmetric and the secondary
substances are optionally asymmetric. To this character-
istic property one should add another which might be called
the "replaceability" or "non-replaceability" of a given
optic isomer by its antipode. Substances possessing ob-
ligatory asymmetry are found in nature in the form of one
only of the two optical isomers, whilst the secondary sub-
stances are found as well in the dextrorotatory as in the
laevorotatory form, often as inactive racemates. We shall
describe this property as exclusiveness or non-exclusive-
ness of the asymmetry-sign.
Exclusiveness of the asymmetry-sign in primary sub-
stances is a well established fact. In amino-acids, no
exception has ever been recorded. Only dextrorotatory
alanine, laevorotatory leucine, dextrorotatory valine,
laevorotatory histidine, laevorotatory aspartic acid, etc.,
have been isolated from animal or plant tissues. All
apparent exceptions to this rule could be traced to some
experimental error as shown by Pringsheim (1910).
The same holds true for the carbohydrates which pos-
sess obligatory asymmetry. Only dextrorotatory glucose,
laevorotatory fructose, etc., can be found in living material.
The isomer which is present in the biological material
is often called "natural," whilst its antipode which is pre-
pared synthetically is considered unnatural, but it is evi-
dent that the term "natural" as a synonym of "biological"
is somewhat improper.
6. N on-Exdusiveness of the Asymmetry-Sign in Sec-
ondary Substances. Turning now to the substances in
which optical purity is not obligatory, we find that one
optical isomer is found in one species of plants and its
antipode in another.
Let us consider first the optionally asymmetric carbohy-
drates. Arabinose, which is found in organic nature in the
racemic state, can also be present in the form of the rela-
tively pure dextrorotatory and of the relatively pure lae-
vorotatory isomers. The left form is the most widely
OPTICAL ACTIVITY OF BIOL. MATEBIAL
29
spread ; it was found, for example, in the leaves of Adonis
verualis (Eken-stein and Blanksma, 1908), entering in the
composition of their glucosides. The right arabinose was
found in the gincoside from Barbados, the so-called barba-
loine (Leger, 1910).
Similar findings were recorded in alkaloids. The laevo-
rotatory alkaloid sparteine, for instance, is widely spread
in plants ; it was repeatedly isolated from Spartium sco-
pariiim and Liipinus liiteus. Recently Orechoff, Eabino-
witch and Konowalowa (1933) discovered the dextrorota-
tory isomer of sparteine in SopJiora pachycarpa, a plant
from Middle Asia.
Blockmann and Roth (1935) reported to have isolated
and obtained in a chemically pure state laevorotatory alca-
nine, a red dye found in the roots of Alkanna tinctoria, a
South-European species ; the dextrorotatory isomer of the
same substance was obtained from the roots of the Japa-
nese plant, Lithospermum erytlirorhizon.
The terpenes were recorded often as dextrorotatory in
one species and laevorotatory in another (Oudin, 1932;
Branke and Parishev, 1937). We tabulated below (Tables
5 and 6) some data on the distribution of the optical
isomers of the two most important terpenes, borneol and
limonene.
It is clear, then, that in secondary substances, both opti-
cal isomers participate in the composition of living sys-
TABLE 5
The Distribution of the Optical Isomers of Borneol in
Different Plants (Bartelt, 1910)
1-Borneol
d-Borneol
Finns maritima (Belloni, 1906)
Amomum cordamomiim (Schimmel,
Thuja occideiitalifi (Wallacli, 1901)
1897)
Andropogon nardus (Schimmel, 1899)
Dryobalanops sp. (Schimmel, 1905)
Asarum canadense (Power and Lees,
Lavandula spica (Bouchardat, 1893)
1902)
Salvia officinalis (Schimmel, 1895)
Blumea hdlsamifera (Haller, 1886)
Fyrctlirum partheniiim (Schimmel,
1894)
Tanacetum vulgare (Schimmel, 1895)
30
OPTICAL ACTIVITY OF BIOL. MATERIAL
TABLE 6
The Distribution of the Optical Isomers of Limonene and of Its
Eacemic Form in Different Plants (Bartelt, 1910)
d-Limonene
1-Limonene
dl-Limonene
(= dipentene)
Pinus serofina
Juniperus virginiana
Andropogon sp.
Laurus campliora
Pittosporum tmdulaium
Canarium sp.
Citrus madurensis
Barosma sp.
Myrrlia electa
Andropogon nardus
Finns silvestris
Andropogon citratus;
A. nardus
Laurus camphora
Citrus madurensis
Barosma sp.
Carum carvi
Foenicidum vulgare
Anethum graveolens
Apimn graveolens
Mentha sp.
Erigeron canadensis
Lindera sericea
Massoia aromatica
Mentha sp.
Foenicidum vulgare
Mentha sp.
Abies pectinata
Monodora myristica
Peumus holdus
Croton eluteria
E ucalyptus staige-
riana
Picea excelsa
Piper nigrum
Myristica officinalis
Lindera sericea
Xanthoxylum sp.
Myrtus communis
Thymus capitatus
Valeriana officinalis
Solidago canadensis
terns, although one of them is usually found more often
than the other.
7. Relative Configuration of Biological Material. The
results of numerous investigations undertaken to establish
the relative configuration of organic substances may be
summarized as follows. All biological isomers of amino-
acids possess the same relative configuration, Fischer and
Raske (1907) observed that from biological (-) serine can
be obtained biological (+) alanine and biological (-) cys-
OPTICAL ACTIVITY OF BIOL. MATERIAL 31
tine. The suggestion that the reUitive configuration in all
biological isomers of amino-acids is identical was made by
Clougli (1918) ; it received confirmation from the work of
a number of later investigators (Freudenberg and Rhino,
1924; Langenbeck, 1925; Karrer and Ehrenstein, 1926;
Levene and Mardaschew, 1937 ; Pfeitf er and Christeleit,
1937). In general, it is established that the primary sub-
stances, although they rotate the plane of polarized light
in different directions, possess the same relative configura-
tion and form a definite "biological series" of optical
isomers. Their antipodes are excluded from participation
in living processes. Not all the potentialities of dissym-
metric configuration, therefore, are employed in the
organization of living systems.
8. Asymmetry as a Criterion of the Organic Origin of
a Substance. From what has been said, it follow^s that
every deviation from the racemic state, that is, every
asymmetry of molecular aggregates represents actually a
specific attribute of biological systems, and we do not know
a single case when it would take place outside of living*
organisms or of the products of their activity. Conse-
quently, optical activity can be used as a criterion of the
biological origin of such natural products as petroleum.
Two theories of the origin of petroleum are generally held,
one attributing it to an inorganic, the other to an organic
source. The first suggestion concerning the inorganic,
volcanic origin of this so-called mineral oil is due to Hum-
boldt (1804). The theory of its organic origin is still
older (Lemery, 1675; Lomonosoff, 1761; Spielmann, 1774).
In its more modern form (see, e.g., Engler, 1906), this
theory implies that the fat contained in the dead bodies of
fishes, molluscs and other sea animals, and especially the
stable palmitic, stearic and oleinic acids are the ancestors
of petroleum. As a result of a breaking down of the chains
of carbon compounds under high pressure, hydrocarbons
with comparatively low boiling point could arise, a poly-
merization of which, during geological periods, resulted in
our present-day oil. It is also possible that in some cases
the initial substance was of vegetable origin.
32 OPTICAL ACTIVITY OF BIOL. MATERIAL
The theory of the organic origin of oil entered a new
phase when Tschngaeff and Walden (1900) pointed ont the
significance of the forgotten observations of Biot (1835)
on the optical activity of oil as a criterion of its origin (see
for further confirmation of these views Vernadsky, 1934).
Since the asymmetry of molecular aggregates and their
optical activity represent an attribute of the material of
living systems only, the theory of the organic origin of oil
can be considered as based on solid ground.
The genesis of the optical activity of oil is far from clear.
Natural fats or glycerides, except lipoids of the lecithin
type and fats with active acid radicals, are optically inac-
tive and do not possess any structural dissymmetry.
Neuberg (1907) outlined the following scheme for the
transformations undergone by these structurally inactive
fats in the process of oil formation. Inactive trioleine,
which constitutes a considerable part of vegetable and
animal fats, would be the original source. By oxidation
or hydration, the structurally inactive free oleinic acid
would be transformed into a dissymmetric racemic body,
for instance, into dioxystearic acid. If now the racemic
trioleine with oxidized or hydratated radicals is subjected
to the asymmetry-producing action of the fat-splitting
enzymes, optically-active fatty acids would arise. Neu-
berg and Rosenberg (1907) performed all these transfor-
mations experimentally; after having obtained optically
active fatty acids out of structurally inactive material they
transformed these active acids into optically active oil.
According to another suggestion of Neuberg (1906), sup-
ported by Trask (1937), the active constituents of oil may
result from the transformation of proteins of dead bodies.
In putrefaction and in autolysis, the transformation of
amino-acids into corresponding fatty acids is possible ;
dextrorotatory isoleucine, for instance, has been trans-
formed into optically active capronic acid. The latter
could, by further condensation, give the numerous optically
active hydrocarbons of oil.
OPTICAL ACTIVITY OF BIOL. MATERIAL 33
SUMMARY
1. Dissymmetric molecules are found in inorganic nature
where they have evidently no relation to life, but it is ques-
tionable whether life is possible without dissymmetric
molecules.
2. In inorganic nature, the two forms of dissymmetric
molecules are always represented in equal concentrations
and the aggregate of molecules thus formed is symmetric
(racemic mixture). 3. Asymmetry of molecular aggre-
gates is a specific property of protoplasm and of living
systems.
4. Primary constituents of protoplasm, such as the
amino-acids, the lecithins and the majority of the impor-
tant sugars are present in protoplasm in the form of only
one of the optical isomers: they are obligatorily asym-
metric. 5. In these substances, the sign of the optical
activity is not replaceable by the opposite sign. 6. The
primary constituents of protoplasm are structurally re-
lated to each other and form ''biological series" of optical
isomers.
7. The secondary constituents of protoplasm which,
functionally, represent storage material or excreta are not
obligatorily asymmetric ; they are sometimes found in liv-
ing organisms in the racemic state. 8. The sign of their
optical activity is replaceable by the opposite sign, so that
one optical isomer is sometimes found in one species and
its antipode in another.
9. The optical activity of mineral oil lends support to
the theory of its organic origin.
BIBLIOGRAPHY
ACKEKMANN, D., Advances in Modern Biol. (Russian), 4, 5, 1935.
BARTELT, K., Abderh. Bioch. Handlex., 7, 266, 1910.
BENDRAT, M., Planta, 7, 508, 1929.
BENNET-CLARK, T., Ann. Rev. Biochem., 6, 579, 1937.
BOYS, S. F., Proc. Eoij. Soc. A.. 144, 655, 1934.
BRANKE, J. AND PARISHEV, J., Plants of the Maritime Region and their
Volatile Oils. Vladivostok (in Russian), 1937.
BROWX, H. AND MORRIS, H., Journ. Chem. Soc, 63, 604, 1893.
CLOUGH, G. W., Journ. Chem. Sor., 113, 526, 1918.
34 OPTICAL ACTIVITY OF BIOL. MATERIAL
EHRLICH, F. AND WENDEL, A., Biochem. Z., 8, 399.
EKENSTEIN van, W. A. and BLANKSMA, J., Chem. Zhl., p. 119, 1908.
ENGLER, C, Chem. Zeitung, 30, 711, 1906.
rA.RBER, E., NORD, F. and NEUBERG, C, Biochem. Z., 112, 313, 1920.
FISCHER, E., Ber. chem. Ges., 40, 1501, 1907.
FISCHER, E. AND RASKE, K., Ber. chem. Ges., 40, 3717, 1907.
FREUDENBERG, K. and RHINO, F., Ber. chem. Ges., 57, 1547, 1924.
GINTL, W. F., Journ. praht. Chem., 106, 489, 1869.
HESS, K. AND WELTZIEN, W., Ber. chem. Ges., 53, 119, 1375, 1920.
JAEGER, F. M., Bee. irav. chim., 3S, 171, 1919.
KARRER, P. and EHRENSTEIN, M., Helvet. chim. Acta, 9, 323, 1926.
KATAGIRI, H. and KATAHARA, K., Bioch. Journ., 31, 909, 1937.
KUHN, W., Ergehn. Ensymforseh., 5, 1, 1936.
, Angew. Chem., 49, 215, 1936.
LANGENBECK, W., Ber. chem. Ges., S8, 227, 1925.
LeGER, E., C. E. Acad. Sc, 150, 983, 1695, 1910.
LEMMLEIN, Trav. Lab. Biogeochimique. Ac. Sc. U.B.S.S., 5, 1938.
LEVENE, P. and MARDASCHEW, Journ. Biol. Chem., 117, 179, 1937.
LIPPMAN, E., Ber. chem. Ges., 17, 2835, 1884.
LOWRY, T. M., Optical Rotatory Power, Longmans, Green & Co., London,
1935.
MAYER, P., Biochem. Z., 1, 39, 1906.
Mckenzie, a. and wood, J., Joum. Chem. Soc., 115, 828, 1919.
MENSHIKOV, G., Bull. Acad. Sc. U.B.S.S., Ser. Chimie, 5, 1035, 1937.
MORISHIMA, K., Arch. exp. Path. Pharmal'., 43, 217, 1900.
NEUBERG, C, Ber. chem. Ges., 33, 2243, 1900.
— , Biochem. Z., 1, 368, 1906; 7, 199, 1907.
NEUBERG, C. and ROSENBERG, E., Biochem. Z., 7, 191, 1907.
ORECHOFF, A., RABTNOWITCH, M. and KONOWALOWA, R., Ber.
chem. Ges., 66, 621, 1933.
OSHIMA, K. and TOLLENS, B., Ber. chem. Ges., 34, 1422, 1901.
OUDIN, A., C. B. Acad. Sc. 105, 185, 1932.
PASTEUR, L., Bev. Scientif. Ill, 4, 2, 1884.
PFEIFFER, P. AND CHRISTELEIT, W., Z. physiol. Chem., 245, 197, 1937.
PRINGSHEIM, H., Z. physiol. Chem., 65, 89 and 96, 1910.
RUHLAND, W., and WETZEL, K., Planta, 7, 503, 1929.
SCHULZE, E. AND WINTERSTEIN, E., Z. physiol. Chem., 45, 79, 1905.
SCHWARZE, P., Planta, 18, 168, 1932.
STOKLASA, J., Z. physiol. Chem., 50, 303; 51, 156, 1907.
THIELE, A., Abderh. Bioch. Handlex., 1, 1149, 1911.
TRASK, P. D., Nature, 140, 857, 1937.
TROMSDORFF, Neue Jahrb. Mineralogie, 1937.
VERNADSKY, W., Problems of Biological Geochemistry, Leningrad (in
Russian), 1934.
WALDBN, P., Nat^irwiss. Bundschau, 15, 145, 1900.
CHAPTER II
THE ORIGIN AND MAINTENANCE OF OPTICAL AC-
TIVITY IN LIVING MATTER
There are, in living matter, some components which con-
stantly prodnce optically active substances while other
components always racemize. The mechanism which in-
sures the constant production of asymmetric substances
needs an explanation. It is somewhat surprising that, for
a long time after optical asymmetry had been recognized
as a characteristic of some constituents of living matter,
the problem of the origin of this asymmetry and of the
maintenance of the optical purity of protoplasm was so
little investigated.
1. The Transmission of the Asymmetric State hy Asym-
metric Synthesis. Before studying how the asymmetric
purity of the primary components of protoplasm is main-
tained, let us consider how the asymmetric state is trans-
mitted from one aggregate of molecules to another or, in
other words, how the asymmetry of protoplasm is "multi-
plied." To answer this question, Pasteur (1860) sug-
gested that every asymmetry owes its existence to some
asymmetric forces operating at the moment at wiiich the
asymmetry appeared. In this manner, one asymmetric
substance would bring into being another in the same way
as life produces life. The principle "Omne viviim ex
vivo" would be paralleled in the transmission of asym-
metry,
Pierre Curie (1894) expressed the same fundamental
principle as follows: "If a phenomenon possesses a defi-
nite asymmetry, the same asymmetry can also be detected
in the causes which have given rise to the phenomenon."
Curie admits that a given asymmetry gives birth to an-
36 ORIGIN AND MAINTEN. OF OPT. ACTIVITY
other asymmetry of the same order of magnitude, i.e.,
possessing the same degree of optical purity. We shall
refer to this view as Curie's principle.
Emil Fischer (1894), after having stated that "one
active molecule gives birth to another," illustrates his
statement by the following concrete example : ' ' The for-
mation of sugar in plants, according to the observations
of physiologists, takes place in chlorophyll grains, which
themselves consist of optically active substances." (The
optical activity of chlorophyll has been demonstrated
recently by Stoll and Wiedemann, 1933.) "I assume,"
continues Fisher, "that, in the formation of sugar, there
is a combination of carbon dioxide or of formaldehyde
with these substances of the chlorophyll grains and that
the subsequent production of sugar proceeds asymmetri-
cally on account of the presence of the asymmetric mole-
cules of chlorophyll." Thus Fischer considered chloro-
phyll to be an asymmetric catalyst, the asymmetric state
of which is transmitted to the molecules of the organic
substance undergoing synthesis and these are, therefore,
represented by only one optical isomer. The notion of
asymmetric synthesis was thus introduced.
Somewhat earlier, Fischer (1890) had proposed another
explanation for the origin of the asymmetric state. He
assumed that, in plants, as in the laboratory, racemic com-
pounds would appear first and that these would subse-
quently be split up by the plant itself into their optical
antipodes.
A few years after Fischer had expressed this opinion,
Brown and Morris (1893), in a thorough study of sugar
metabolism in plants, could find in them neither racemic
nor laevorotatory glucose. This finding caused Fischer
to abandon his previous idea.
It is now well known that laevorotatory glucose does not
occur in living organisms, that it practically does not fer-
ment and that it is not used as food by plants or animals.
Furthermore, a great deal of experimental data has been
accumulated which shows that, in different enzymatic re-
ORIGIN AND MAINTEN. OF OPT. ACTIVITY 37
actions, no intermediate racemic glucose is formed, but the
optically active product is obtained immediately (c/.,
Tomivasu, 1937).
The catalytic transmission of the asymmetric state was
later considered by Strong (1898) in a well known discus-
sion on asymmetry and vitalism.
Asymmetric syntheses were soon experimentally real-
ized in the laboratory. Marckwald (1904) synthesized
optically active valerianic acid from structurally inactive
methylethylmalonic acid in the presence of active brucine.
He defined asymmetric synthesis as a process "in which
optically active substances are obtained from symmetric
compounds through the intermediary of optically active
substances."
The same year McKenzie also realized some asym-
metric syntheses.
Since these pioneer investigations, the literature on this
subject has expanded considerably and the synthesis of
optically active compounds from structurally inactive
material has been carried out by a number of other chem-
ists. These researches have been well reviewed by Mc-
Kenzie (1932, 1936) and by Ritchie (1933) to whom we
refer the reader.
But, on the question of the fundamental physical mecha-
nism by which the asymmetric state of the catalyst is
transmitted to the substance acted upon, there are only
some still incompletely shaped theories, for example, the
theory of the so-called asymmetric induction (see Ritchie,
1933)*.
To summarize, the authors whose views have been de-
scribed in this section admitted, generally, that some opti-
cally active, relatively simple compounds appeared once
in nature and that, bv asvmmetric syntheses, the asvm-
metric state has been transmitted to other compounds
more and more complicated in structure.
2. The Transmission of Asymmetry , from the Thermo-
dynamic and Kinetic Point of Vieiv. Recent investiga-
tions of the kinetics of asymmetric synthesis have con-
38
ORIGIN AND MAINTEN. OF OPT. ACTIVITY
siclerably modified our ideas concerning the maintenance
of the asymmetric state.
Among the first observations on this subject, one should
mention those of Bredig and Fajans (1908) and those of
Fajans (1910) on the asymmetric splitting of racemic
camphorocarbonic acid into camphor and carbonic acid in
the presence of various catalysts.
Almost simultaneously, Rosenthaler (1908) began his
studies on the asymmetric synthesis of the nitrite of man-
delic acid, which were later on repeated and extended by
a number of other investigators and which constitute, at
present, the basis for the general theory of asymmetric
synthesis. He observed that, by combining the symmet-
ric molecule of benzaldehyde with the symmetric molecule
of hydrocyanic acid under the action of the asymmetric
catalyst emulsine, one obtains an optically active nit rile
of mandelic acid. A considerable excess of dextrorotatory
over laevorotatory nitrite was recorded. Rosenthaler
also pointed out that the optical activity of the product
synthesized by emulsine reached a maximum value after
a certain time and then decreased (r/. Table 7 ).
TABLE 7
Change in Optical Activity during the Enzymatic Synthesis of the
NiTRiLE OF Mandelic Acid (Eosenthaler, 1908)
(The numbers give the optical rotation of the synthetic product)
Temperature
Time from the beginning of the synthesis :
1 hour
3 hours
24 hours
25° C.
30° C.
1.8
2.2
2.8
2.6
2.0
2.1
Other observations on the change of optical activity dur-
ing asymmetric synthesis were made by Nordefeldt
(1922). The optical activity was found to tend asympto-
tically to zero {cf. Fig. 3).
Bayliss (1913), Krieble (1913) and Nordefeldt (1922)
showed the important fact that the synthesis of racemic
mandelo-nitrile takes place in the absence of enzymes and
ORIGIN AXD MAINTEN. OF OFT. ACTIVITY
39
>-
>
H
O
<
_J
<
o
I-
Q.
O
TIME — *■
Fig. 3. Change in optical activity, as a function of time, in the enzymatic
asymmetric synthesis of the nitrile of mandelic acid (from Kulm, 1936).
that the addition of the latter accelerates the reaction and
gives to it an asymmetric character but does not influence
its equilibrium constant.
These observations made it possible for Werner Kuhn
(1936) to undertake the theoretical analysis of the prin-
ciples of the asymmetric synthetic action of enzymes. We
shall summarize here his more important conclusions.
It should be pointed out, first, that the separated active
components of a given organic substance and the equi-
molecular mixtures of these components (racemates) are
not equivalent from the thermodynamic point of view.
The mixing of the components into a racemate liberates
energy, while their separation requires an expenditure of
work. Consequently the optically active state is not a
state of equilibrium as compared to the racemic state.
The question arises, then, as to the manner in which such
conditions of thermodynamic disequilibrium can be real-
ized in catalytic reactions in living matter. One might
first inquire whether such reactions are true catalytic
reactions or not.
Let us consider the characters of a true catalysis lead-
ing to the formation of an asymmetric compound. Inas-
much as the preparation of the left and that of the right
antipode of a given substance in equal concentrations are
equivalent from the standpoint of energy expenditure, the
40 ORIGIN AND MAINTEN. OF OPT. ACTIVITY
constant Ki of equilibrium between the /-antipode of the fi-
nal substance and the initial product must be equal to the
constant K^ of equilibrium between the c/-antipode of the
final substance and the initial product. If c is the con-
centration of the initial substance, c'l the concentration
of the 7-antipode and c'a the concentration of the d-m\ti-
pode of the final substance, one has
c'l/c = K, = c'd/c = Kd (1)
Condition (1) characterizes a true catalysis. If this con-
dition is not fulfilled, the initial substance will be simul-
taneously in equilibrium with different concentrations c'l
and c\i of the two antipodes and the final product will be
partially optically active. But this is thermodynamically
impossible in the case of true catalysis.
Another character of true catalysis is that the value of
the equilibrium constant in equation (1) is the same ir-
respective of whether a catalyst is used or not. The
velocity constant A-,, in the formation of the f/-antipode
from the initial material and the velocity constant k'^ in
the reverse conversion are increased by the catalyst to
the same degree. If the addition of an enzyme would in-
fluence the two velocity constants differently and change
the equilibrium constant, the reaction would not be a true
catalysis and the final product would be optically active.
Experimentally, as has been said above, it was found
that, in the synthesis of the nitrile of mandelic acid, the
use of the catalyst does not change the equilibrium
constant.
That both velocities I'a and k'a are accelerated to the
same extent by the catalyst has been proved in optically
non-specific enzymatic reactions {cf. Borsook, 1935).
Furthermore, Nordefeldt (1922) has observed that if in
the synthesis of mandelo-nitrile, one adds emulsine when
the reaction has already proceeded for a while, the enzyme
does not change anything in that which has already been
transformed, it exerts its asymmetrical effect only on the
material yet to be transformed. In a system which has
reached the state of equilibrium without enzymes, the ad-
ORIGIN AND MAINTEN. OF OPT. ACTIVITY 41
ditioii of an enzyme does not change anything either.
This does not leave any doubt that, in the case of these
isolated enzymatic transformations, we are dealing with
true catalysis.
Finalh% in true catalysis, the optical activity of the sub-
stance being formed represents only a temporary phe-
nomenon which gradually disappears. This will be clearer
when we have examined the dynamics of the two ways in
which optical activity could be obtained in biochemical re-
actions, namely, the splitting up of racemates and asym-
metric synthesis.
In the splitting of a racemate consisting of two anti-
podes, Ai and A^, which change respectively into Bi and
B,j, one can represent the process as follows:
k.
k'
(2)
If the left initial product Ai is transformed into Bi with
a velocity constant A,, different from the constant A-^ with
which Aa is transformed into B^, there results optical
activity. If Jii = Jici the racemate will be split up sym-
metrically.
In asymmetric synthesis, a symmetric initial substance
A is transformed with different velocities, A^^ and k^, into,
respectively, Bi and B,„ according to the diagram
(3)
A-
Kuhn integrated the systems of differential equations
42 ORIGIN AND MAINTEN. OF OPT. ACTIVITY
corresponding to these two cases and studied the dynamics
of the change of optical activity in terms of time.
In the case of the splitting up of a racemate (2), if
ki/k,, >> J , and K >> 1, the substance Bj will be obtained
almost exclusively at the beginning; its concentration
might approach c„/2 (if c,, is the concentration of the initial
substance) ; later, A^, will be transforming itself into B,i
till, finally, the concentrations Bj and Ba are equalized. At
the initial and final states the solutions will be optically
inactive.
In the case of asymmetric synthesis (3), assuming again
that ki/ka >> 1 and K >> 1, there will be, at the begin-
ning, an accumulation of the /-form, B,-, the whole initial
material (c,,) will be practically transformed into this
/-antipode, since the velocity constant A",, is supposed to be
very low as compared to k,. The concentration of the initial
substance A will approach cjK. A will also change very
slowly into B,j and, as a result of this change, its concentra-
tion will be reduced and the equilibrium between A and Bi
will be disturbed: consequently, a certain quantity of B,
will be transformed into A. This will cause a further
transformation of the initial substance into B,i. The pro-
cess will continue as long as the initial substance A is in
equilibrium simultaneously with Bj and B,i or, in other
words, until the racemic state is obtained. So the same
catalyst which, at first, brought about the transformation
of A into practically pure antipode B, later causes a com-
plete racemization of the product.
It is of interest to inquire what is the difference in the
stability of the temporary state of optical activity in the
case of the splitting up of a racemate and in that of asym-
metric synthesis. Kuhn showed that the ratio H between
the time T^ necessary for racemization and the time Ti
necessary for the attainment of maximal activity is
H = ki/ka ■ K/2 in the case of asymmetric synthesis and
H = ki/kf, in the case of the splitting of the racemate. The
factor K/2 is absent in the second equation. Since the
constant K is large, it is evident that the stability of the
ORIGIN AND MAINTEN. OF OPT. ACTIVITY 43
optically active state will l)e considerably greater in
asymmetric synthesis than in the splitting of racemates.
The experimental data reported above, concerning the
temporary character of optical activity {cf. also Bredig
and Fajaiis, 1908; Bredig and Minaeft", 1932; Nordefeldt,
1922) are in agreement with. Knhn's calculations. The
conclusion to derive from this agreement is that
true catalysis occurs in the isolated enzymatic systems
considered.
Furthermore, it should be noticed that asymmetric syn-
thesis, which seems to take place in protoplasm rather
than dissociation of racemates, is precisely the process
which secures a longer duration of the state of optical
activity.
The maintenance of asymmetry in mineral oils is
probably to be explained by the extreme slowness of the
transformations which take place in them.
3. Maintenance of Optical Purity by the So-C ailed
" Stereo-aiitonomic Substances." If, in enzymatic sys-
tems, one has to do with true catalysis, and in true
catalysis there is a gradual decrease in optical activity,
one might expect that a substance formed in an asym-
metric synthesis be optically less pure than the compound
from which it originates (Langenbeck and Triem, 1936).
Curie's principle, postulating that any asymmetry origin-
ates from another asymmetry of the same order would
not hold then. So the presence of protoplasmic com-
ponents in the form of pure optical isomers for an in-
detinitely long time still lacks an explanation. The
features which, in the organization of protoplasm, are
responsible for the maintenance of optical purity are still
to be found.
Kuhn (1936) showed that, in some cases, the mainten-
ance of optical purity in a system, despite the gradual
decrease of optical activity in a single synthetic enzymatic
process, can be explained by the behavior of some sub-
stances that he called "stereo-autonomic." It is knowni
that the right nitrile of mandelic acid, when synthesized
44 ORIGIN AND MAINTEN. OF OPT. ACTIVITY
by the plant, is stored not as such but combined with
gentiobiose in the form of 3-giucoside (natural amyg'da-
line). The latter easily crystallizes from water solutions,
while the glucoside which consists of gentiobiose and of
the left nitrite of mandelic acid possesses such a high
solubility that it does not, in general, crystallize from
water solutions (Walker and Krieble, 1909; Krieble,
1912).^ So, the fact that pure natural amygdaline is de-
posited in the plant does not necessarily postulate the
existence of an optically specific enzyme, synthesizing only
the right nitrite of mandelic acid. The right and the left
nitrites may be produced; then the right component pre-
cipitates in the form of gentiobioside ; the excess left
nitrile can thereupon be racemized according to the re-
quirements of true catalysis ; the right nitrile originating
from this process is again bound to gentiobiose and the
process continues until all the nitrile is converted into the
less soluble gentiobioside of the right nitrile, i.e., into pure
natural amygdaline. The same final state would evi-
dently ensue no matter whether the enzyme possesses the
capacity of preferential synthesis of the right nitrile or if
it would synthesize racemic nitrile. In the latter case,
however, the gradual catalytic transformation of the left
component into the initial substance (benzaldehyde and
hydrocyanic acid) and the resynthesis of the right com-
ponent would demand a long time, which is evidently
spared by the utilization of an optically specific enzyme.
Natural optically active gentiobiose is a stereo-autonomic
substance in the sense that it conditions the stable optical
purity of the synthetic product. Kuhn sees a confirmation
of his views in the fact that, in the fruits of Prunns
laurocerasus, one finds a gentiobioside of the pure right
nitrile of mandelic acid, while, in the leaves of the same
plant, one finds a glucoside of the racemic nitrile. It is
probable that the difference in solubility brought about
1 Similar differences are found, in general, in diastereomers, that is, in
substances consisting of one antipode of a substance A combined Avitli either
of the tAvo antipodes of a substance B. For example, AiBi and AiBa are
two diastereomers.
ORIGIN AND MAINTEN. OF OPT. ACTIVITY 45
by glucose as a component of the glucoside and by gen-
tiobiose as a component of the gentiobioside is responsible
for the fact that they are fomid in nature as indicated.
4. Procedures Used by Nature for Mainfaiuing Optical
Purity and Establishing a ''Fixed Internal Milieu." The
evolution of living beings has consisted in a gradual in-
crease of the number of fixed parameters of the internal
milieu. For example, in the transition from poikilother-
mic into homoiothermic animals, the body temperature has
been fixed at a constant value. The non-dependence on
the temperature of the external medium has given the
homoiotherms important advantages over the cold-blooded
animals in natural selection.
Considering such cases of specific fixity acquired by the
internal medium, Claude Bernard made his famous state-
ment : * ' La fixite du milieu interieur est la condition de la
vie libre " ( " The fixity of the internal medium is the con-
dition of independent living").
The elaboration of mechanisms in living matter to
maintain optical purity evidently contributes to the fixity
of the internal milieu. The spatial parameters which de-
termine the asymmetry of a substance are fixed in primary
constituents of protoplasm in such a way that optical
purity is maintained.
Barcroft (1934) notes that two methods are used by
nature to secure the constancy of internal medium, the
method of evasion and the method of correction.
a) Widely Different Velocities in the
Formation of the Two Optical Iso-
mers. As a mechanism of evasion nature uses a high
ratio of the reaction velocities. A-,/ A',,. While, in the cases
of catalysis more commonly encountered, this ratio is of
the order of 1 to 2, in enzymatic reactions the ratio reaches
100, 1000, or even greater values. This ratio evidently
determines the degree of predominance of the right or of
the left isomer in the substance synthesized. A great dif-
ference between the velocity constants, Jii and k,i, makes that
46 ORIGIN AND MAINTEN. OF OPT. ACTIVITY
the non-iitilizable isomer appears only in insignificant con-
centrations in the first stages of synthesis. A large value
of the constant of equilibrium K, on the other hand, as-
sures a more lasting stability of the active state. Both
these factors contribute toward having the system
"evade" for a time the effects of inevitable racemization.
h ) Langenbeck and Triem's IVIechan-
i s m . Recently, Langenbeck and Triem (1936) have
shown that an increase of optical purity can be obtained
in interrupted reactions between two optically impure
substances. If an optically impure enzyme is acting upon
an optically impure substance and if the reaction is not
allowed to proceed to the end, the optical purity of the
system may be increased. Let us suppose that two sub-
stances, optically active but not optically pure, A and B,
combine to form AB. Let us assume also that the
laevorotatory isomers, Ai and Bi, predominate over their
antipodes. A,, and B^. The following partial reactions
will take place
A. + B,->A,B.
A, + B, ^ A,B,
A, + Ba -^ A.Ba
A, + B,^A,Bi
Since [A.] > [AJ and [B,] > [BJ, we shall have, if
we interrupt the reaction before it is completed,
1A3J [AJ [A.B,] rB.]
[A,B,] ^ [AJ "^'^^ [A,BJ ^ [BJ '
If, for instance, the concentrations of the initial sub-
stances, Ai and A,,, are in the ratio 2 : 1, and if the concen-
trations of Bi and B,, are identical respectively to those of
Ai and A,,, i.e., are also in the ratio 2: 1, a time will come
at which the ratio of the concentrations of the enantio-
morphic products, AjB, and A^B,,, will be the product of
2 X 2
the ratios, tttTj that is, 4 : 1. If the reaction is inter-
rupted at that time, the optical purity of the transformed
material will be increased. (It is certain that, simultan-
ORIGIN AND MAINTEN. OF OPT. ACTIYITY 47
eoiislv, the remainder of the untranst'ormed substances
will undergo a" corresponding decrease in optical purity.)
Langenbeck and Triem (1936) proved experimentally
that the optical activity can be increased in reactions of
this type. They synthesized /-tyrosine anhydride from
/-tyrosine methyl ether and observed a concentration of
30.8% of /-tyrosine in the final product while the initial
substance contained only 27.4%.
It is possible that such processes have taken place in
the enzymatic origin of ferments, that is, when one fer-
ment has been synthesized wdth the aid of another opti-
cally active ferment. Then the necessary decrease of
optical purity of the initial material is of no importance
since only the newly formed ferment, in the interrupted
reaction, will transmit to some other substance its
increased optical purity.
It should be noted, in relation with the reactions
described in this section, that the succession of synthetic
processes which take place continuously in living systems
might in itself be an important factor in the evasion of
the effects of racemization. The incessant reconstruction
of living matter should then, perhaps, be considered as
an indispensible condition for the maintenance of the
optical purity of stereo-autonomic substances.
It is usually thought that, though nature might evade
for a time the eifects of racemization, finally the latter will
inevitably set in and that nature does not possess any
method of correction by which it would remove the un-
natural isomer and actively resist racemization. Kuhn
(1936) not only accepted the idea of the absence of such
active resistance, there being no enzyme know^n for per-
forming this function, but he thought that the racemiza-
tion which finally takes place might constitute, in part, the
process of ageing.
The fact that, when animals and plants are fed with
racemic amino-acids, they principally consume the natural
isomers of the left steric series and leave the other isomer
intact, has been, in general, considered as proving that the
48 ORIGIN AND MAINTEN. OF OPT. ACTIVITY
organisms are devoid of enzymes suitable for catalyzing
transformations of the unnatural isomers.
Scliulze and Bosshard observed this selective action of
one isomer in lower organisms already in 1886 and their
data were later confirmed by a great number of authors
and especially by Pringsheim (1910).
Many similar observations were made on mammals. A
dog which receives a racemic preparation of leucine
(Abderhalden and Samuely, 1906) or of alanine (Abder-
halden and Schittenhelm, 1907) consumes preferably the
natural isomers and excretes in its urine a large portion
of the unnatural amino-acids. The same was observed
later by Abderhalden and Tetzner (1935) in rats, rabbits
and dogs fed with racemic alanine.
But another series of facts points out the possibility of
the presence, in living protoplasm, of an active mechanism
contributing, by a method of correction, toward maintain-
ing optical purity and thus toward securing the fixity of
the internal medium.
(c) Krebs' Mechanism. Krebs (1933) who has
undertaken extensive investigations on the oxidative de-
amination of different amino-acids by tissue slices of liver
and kidney from rat, pig, cat, dog and rabbit, discovered
the very important fact that, while both optical isomers of
amino-acids are deaminated, the unnatural forms of the
right steric series are almost always deaminated much
more rapidly than the natural ones {cf. Table 8).
These observations were soon confirmed by Kisch (1935)
and by Neber (1936). Some data of Kisch are given in
Table's.
Krebs (1935) assumes that there are two different
enzymatic systems one of which catalyzes the deamination
of the right and the other that of the left amino-acids.
This assumption follows, in particular, from the fact that
the deamiiiation of the left amino-acids is inhibited by
octyl alcohol, Avhile that of the unnatural isomer of the
right series is not affected by octyl alcohol of the same
concentration. He further points out that the data con-
ORIGIN AND MAINTEN. OF OPT. ArTIVITY
49
TABLE 8
Deamination of Optically Active M/20 Amino-acids by Slices
OF Rat Kidney (Krebs, 1933)
Aiiiino-;u'id
ml. of ammonia
Ratio of the velocity
of deamination of
the unnatural to
mg. of tissue x hours
that of the
natural
isomer
i(+)al;niiiie
r7(-)al;inine
Z(+) valine
d(-) valine
I (-) loiu'ine
d (+) leucine
?(-)phenyl-a]amne
d (+) phenyl-alanine
i(— )histidine
d(+)histi(line
3.36 ]
37.80 ^
3.86 ]
57.60 ^
6.68 ]
34.90 1
10.4 ]
77.0 (
3.18]
9.75 (
11.3
14.9
5.2
7.4
3.1
TABLE 9
DEAiriNATioN OF M/50 Amino-acids by Slices of Liver axd Kidney
OF Different Animals (Kisch, 1935)
(The velocity of deamination is expressed in mI/5000 of NH3 per
gram of fresh weight of tissue in 2 hours)
Animal
Organ
Number
of
experi-
ments
Amino-acid
Velocity of deami-
nation of the
natural
isomer
0)
unnatural
isomer
(d)
Ratio of the
velocity of
deamination
of the un-
natural to
that of the
natural
isomer
Rat
Kidney
3
Alanine
15.7
56.8
3.6
1 1
1 1
3
Leucine
3.9
115.6
29.6
i I
1 1
2
Isoleucine
16.2
141.1
8.7
1 1
Liver
2
Alanine
1.9
11.2
5.9
Sheep
Kidney
2
Alanine
13.5
81.6
6.0
< <
< t
2
Leucine
1.2
24.0
20.0
( (
I i
2
Isoleucine
13.2
118.9
9.0
Pig
Kidnev
2
Alanine
16.9
122.2
7.2
( (
i I
0
Leucine
2.8
52.0
18.6
(<
1 1
4
Isoleucine
12.5
136.5
10.9
( (
Liver
5
Alanine
7.1
23.3
3.3
50 ORIGIN AND MAINTEN. OF OPT. ACTIVITY
cerning the oxidative deamiiiatioii do not contradict the
fact of a more rapid consumption by the whole organism
of natural isomers of amino-acids. Natural amino-acids
can evidently be consumed without deamination ; consump-
tion and deamination need not to coincide.
From the standpoint of the problem of the maintenance
of optical purity in protoplasm, Krebs' results are very
significant. The study of catalysis has shown that in
protoplasm composed of optically pure left isomers of
amino-acids the appearance of small quantities of the
right forms is inevitable. Krebs' data suggest that the
organisms have developed a mechanism for removing the
isomers of unsuitable spatial configuration. This mecha-
nism would consist in a deamination of the inappropriate
right forms as soon as they appear. The right isomers
would be transformed into structurally inactive keto-acids
identical with those which can be obtained from the left
amino acids. In this manner, the organisms would by no
means be so helpless in regulating the optical purity of
their protoplasm as was assumed by Kuhn and they would
possess an active method of correction for securing the
fixity of their internal medium.
It is to be noticed that Ritchie (1933), before any of the
researches that we mentioned on oxidative deamination
had been made, admitted a priori the possibility of the
existence of such a method of correction. He wrote that,
while one of the antipodes participates in cell metabolism,
the other, which is formed simultaneously but at a much
lower rate, almost certainly is removed as soon as it is
formed. Ages of evolution would be responsible, accord-
ing to him, for the development of such a physiological
regulating system.
One might, at first, be inclined to consider the existence
of a special enzymatic system acting on unnatural isomers
of amino-acids, as a chance happening without particular
significance. But, then, what sense is there in talking of a
"specificity" of any enzymatic reactions, and in explain-
ing this specificity as a result of a long process of natural
selection? {Cf., Eric Holmes, 1937.)
ORIGIN AND MAI NT EN. OF OPT. ACTIVITY 51
Krebs (11)36) proposed another interpretation of his
data. Following Emil Fischer's somewhat archaic views
on a possible synthesis in protoplasm of racemic amino-
acids and of their subsequent splitting into optical iso-
mers, he considered the deamination by a deaminase
specific for right amino-acids as a process by which the
organism decomposes the racemate and obtains the left
amino-acids required. But it has been seen above that the
experimental data available do not speak in favor of the
hypothesis of a primary symmetric synthesis.
5, Biological Advantages of Optical Purity. In the
study of the methods used by nature to maintain optical
purity, some authors have considered the advantages for
living organisms of working with asymmetric material.
Mills (1932) has attempted to show that living systems
consisting of substances in the asymmetric state are more
efficient than their hypothetical racemic competitors would
be. On the basis of what is known on the stereo-specificity
of the action of, for example invertase, in the hydrolysis
of saccharose one can expect that the common invertase
activate onlv the clextrorotatorv saccharose and that the
optical isomer of this invertase act only on the left saccha-
rose. Let us consider the initial stage of the reaction,
when, with small concentrations of saccharose, the velocity
of hydrolysis is approximately proportional to the con-
centration of the enzyme and to the concentration of the
substance acted upon. In an experiment with optically
pure saccharose and corresponding invertase, every mole-
cule of saccharose coming in contact with the enzyme will
be subject to activation, while in an experiment with a
racemic saccharose and a r//-invertase only one-half of the
collisions will be effective. Consequently, the reaction in
the racemate will take place at a considerably lower rate
than that in the optically active system.
It should be noticed that, in the case of two enantio-
morphic systems of transformations working side by side
(racemic material), the velocities of many processes might
be decreased when the two dissvmmetric substances inter-
52 ORIGIN AND MAINTEN. OF OPT. ACTIVITY
act. Consequently, the synthesis of the components of
new tissues and the growth of the latter will proceed more
rapidly with asymmetric than with racemic material.
If the fundamental physiological processes are more
intense in asymmetric systems, the passage from racemic
to optically active protoplasm was a significant physio-
logical advance which contributed to the survival of asym-
metric protoplasm in the process of natural selection. Be-
sides, the development of asymmetry, by contributing to
the fixity of the internal medium, increased the possibility
of independent life for any given organism, in the sense
of Claude Bernard.
6. The Origin of the Asymmetry of Protoplasm. Assum-
ing that the asymmetry of protoplasm is maintained by
some mechanism devised by nature, a fundamental prob-
lem still remains to be solved, that of the origin of the
initial inequality of the right and the left components of
protoplasm.
In the study of the causes of the initial asymmetry, the
authors have followed two directions. Some have at-
tempted to correlate the origin of the asymmetry of
matter with the asymmetric influence of terrestrial mag-
netism; others have considered asymmetry as originating
in a deviation from a statistical average.
Since C^otton (1896) had shown that solutions of opti-
cally active substances possess different coefficients of
absorption for the right and the left circularly polarized
light, it has been thought that the action of such light
might furnish a promising method of obtaining active
compounds from racemic ones.
It is known that the circularly polarized light is found
in nature, for instance, when the plane-polarized light
from the sky is reflected on the surface of the sea. Byk
(1904) suggested that, because of the rotation of the plane
of polarization of light by terrestrial magnetism, there
must be, in the total quantity of light circularly polarized
at the surface of the earth, a predominance of one of the
two forms of light. This predominant form acting for
ORIGIN AXD MAINTEN. OF OPT. ACTIVITY 53
long' periods of time on racemic compounds would initiate
optical activity.
More recently Kuhn and Braun (1929) and Kulni and
Knopf (1930) have shown that, in laboratory experiments,
when circularly polarized light is used in the photochemi-
cal decomposition of racemates, it causes the appearance
of optically active isomers.
Ritchie (1933) and later Langenbeck and Triem (1936)
supported the hypothesis just described.
The second explanation of the origin of optical asym-
metry (cf., Pearson, 1898; Fitzgerald, 1898; Bartrum,
1898; Errera, 1898; Kipping and Pope, 1898; Byk, 1925;
Mills, 1932) is based on the assumption that the equality
of the right and left components represents a statistical
mean value around which fluctuations occur. Kipping and
Pope (1898) observed, for example, that, while the occur-
rence of either right or left component, in crystallization
experiments, furnished a mean value of 50.08% ± 0.11,
the proportion varied from 24.14/^ to 77.36% in separate
experiments (46 of them). An inequality of the right or
the left form of a substance might have originated acci-
dentally in this manner when some living systems were in
formation and this inequality might have spread by asym-
metric catalysis (Strong, 1898).
Lately Spiers (1937) supported the chance deviation
hypothesis of the origin of asymmetry.
7. General Survey of the Problem of the Origin and
Maintenance of Optical Asymmetry. The various stages
in the development and maintenance of the asymmetric
state are represented diagramatically in Fig. 4.
Let us note, first, that there are two levels of stability
for the state of symmetry or asymmetry: 1. the level of
thermodynamic stability which characterizes the racemic
state ; 2. the level of protoplasmic stability which is main-
tained by living matter. In inorganic nature, the race-
mates are stable because they possess the least amount of
free energy. In living nature, optically pure forms are
stable because they are the most advantageous in natural
54 ORIGIN AND MAINTEN. OF OPT. ACTIVITY
t
>-
>
I-
o
<
<
o
I-
Q.
O
<
I-
a
D
LEVEL OF PROTOPLASMIC STABILITY
c\ \' \ A
■f
B
LEVEL OF THERMODYNAMIC STABILITY
TIME
Fig. 4. Diagram representing the processes involved in the origin and
maintenance of optical purity in living matter. The arrows A represent the
Langenbeck and Triem mechanism; the upward arrows B, the Krebs mecha-
nism; the downward arrows C, the Kuhn mechanism; the horizontal arrow
3 illustrates Curie 's view according to which asymmetry is simply transmitted
from one asymmetric molecule to another.
selection. So the racemic state is stable in inorganic
nature and unstable in living matter.
For living systems to pass from the level of thermo-
dynamic to that of protoplasmic stability and to stay at
the latter level requires a series of mechanisms of which
two have been described : the Langenbeck and Triem
mechanism by which the optical purity is increased in a
series of interrupted reactions (upward arrows in plain
lines, A^ in the diagram) and the Krebs mechanism by
which the unnatural optical isomers are removed (upward
arrows in dotted lines, B).
Since the racemic state represents a state of thermo-
dynamic equilibrium, the initial asymmetry will have a
tendency to disappear (Kuhn's mechanism, downward
arrows, C, in the diagram).
The eifect of circularly polarized light in inducing some
asymmetry will probably not be sufficient to maintain the
high degree of optical purity exhibited by protoplasm.
It is possible also that the Langenbeck and Triem
mechanism, which probably is not efficient enough to main-
tain the almost absolute purity of protoplasm as we know
ORIGIN AND MAINTEN. OF OPT. ACTIVITY 55
it at the present time, was involved in the early stages of
natural selection while Krebs' mechanism, which is more
higlily efficient, was developed only later in evolution.
The asymmetric state of protoplasm and its mainte-
nance by regulative mechanisms appear, then, as a heri-
tage of countless ages of transformations, and as a result
of the elaboration by nature of systems which seem to tend
to some sort of physiological perfection.
SUMMARY
1. According to the earlier authors, asymmetry, once
originated, has been transmitted from one substance to
another, as life is transmitted from one living being to
another.
2. Emil Fischer suggested that asymmetric catalysts
synthesize asymmetric compounds from symmetric ones.
Such asymmetric syntheses were soon realized in labora-
tory experiments.
3. It was then observed that, in an asymmetric synthe-
sis, the optical activity reaches a maximum and then
decreases, and that the enzyme does not influence the equi-
librium constant of the reaction. These observations have
been the basis of theoretical investigations by Kuhn on
the thermodynamics of asymmetric synthesis.
4. Kuhn pointed out that the separation of two optical
isomers requires an expenditure of work, while their mix-
ing into a racemate liberates energy, the optically active
state being a state of disequilibrium. He further showed
that the characters presented by enzymatic reactions are
those thermodynamically expected in true catalysis.
5. Optical purity might be conditioned in some cases by
the behaviour of "stereo-autonomic substances," i.e., of
substances whose properties, such as solubility, maintain
one isomer in solution while the other separates out.
6. To maintain the state of disequilibrium inherent in
optical purity, nature, it seems, has developed regulating-
mechanisms, such as: {a) The use of widely different
velocities in the formation of the two optical isomers in
56 ORIGIN AND MAINTEN. OF OPT. ACTIVITY
asymmetric syntheses; (b) The succession of reactions
which are interrupted before the optical activity has had
time to disappear (Langenbeck and Triem's mechanism) ;
(c) The more rapid deamination of the unnatural isomer
which then separates out (Krebs' mechanism).
7. Optical purity seems to impart to protoplasm some
advantages in natural selection, in particular, it seems to
increase the reaction rate and the growth activity. So, the
establishment of a state of optical purity can be con-
sidered as a method used by nature to stabilize the inter-
nal milieu.
8. The origin of asymmetry has been ascribed by
several authors to the influence, on some reactions, of
circularly polarized light, which would be predominantly
right or left on account of its rotation by terrestrial mag-
netism ; other authors think that the equality of right and
left isomers is a statistical mean value and that the
inequality resulted from fluctuations from the mean.
BIBLIOGRAPHY
ABDEEHALDEN, E. and SAMUELY, F., Z. physiol. Chem., 47, 346, 1906.
ABDEEHALDEN, E. and SCHTTTENHELM, A., Z. physiol. Chem., 61,
323, 1907.
ABDEEHALDEN, E. and TETZNEE, E., Z. physiol. Chem., 232, 79, 1935.
BAECEOFT, J., Features in the Architecture of Physiological Functions,
Cambridge, 1934.
BAETEUM, C. O., Nature, 58, 545, 1898.
BAYLISS, W., Journ. Physiol., 46, 236, 1913.
BOESOOK, H., Ergebn. Ensymforsch.. 4, 1, 1935.
BEEDIG, G. and FAJANS, K., Ber. chem. Ges., 41, 752, 1908.
BEEDIG, G. and MINAEFF, M., Biochem. Z., 249, 241, 1932.
BEOWN, H. and MOEEIS, H., Journ. Chem. Soc., 63, 604, 1893.
BYK, A., Z. physil-al. Chem., 49, 641, 1904.
, NaUirwiss., 13, 17, 1925.
COTTON, A., Ann. Chim. et Phys., 8, 347, 1896.
CUEIE, P., J. de Physique, 3, 393, 1894.
EEEEEA, G., Nature, 68, 616, 1898.
FAJANS, K., Z. phy.nlcal. Chem., 73, 25, 1910.
FISCHEE, E., Ber. chem. Ges., 23, 2138, 1890 ; -21, 3189, 1894.
FITZGEEALD, G. F., Nature, 68, 545; 69, 76, 1898.
HOLMES, E., The Metabolism of Living Tissues. Cambridge, 1937.
JAPP, F. E., Nature, 68, 452, 1898.
KIPPING, F. S. and POPE, W. J., Nature, 69, 53, 1898.
ORIGIX AXD MAIXTEN. OF OPT. ACTIVITY 57
KISCH, B., Biochcm. /., JSO. 41, l!tH5.
KEEBR, H. A., Z. i)hysiol. Clicm., 217, 191, 1938.
, Bioch'. Jom-n., 29, 1620, 1935.
, Ann. Mev. Biochem., 5, 247, 1936.
KEIEBLE, v., Journ. Am<r. Chcm. Soc, 34, 71(>, 1912; 35, 1643, 1913.
KUHN, W., Ergehn. Enziimforscli., 5, 1, 1936.
, Angetc. Chem., 49, 215, 1936.
KUHN, W. and BRAUN, E., Xatiirwisn., 17, 227, 1929.
KUHN, W. and KNOPF, E., Xaturwi.s.s., 18, 183, 1930.
LANGENBECK, W. and TRIEM, G., Z. pluisil-al. Chem., 177, 401, 1936.
MARCKWALD, ^\ ., Bcr. chcm. Gcs., 37, 349, 1904.
McKENZIE, A., J. Chcm. Soc, S'5, 1249, 1904.
— ■, Angew. Chem., 4.5, 59, 1932.
• — , Ergcbn. Enzymforsch., 5, 49, 1936.
MILLS, W. H., Journ. Soc. Chem. Industry, 51, 750, 1932.
NEBER, M., Z. physio}. Chem., 240, 59, 1936.
NORDEFELDT, E., Biochem. Z., 131, 390, 1922.
PASTEUR, L., Reclierches sur la dissymetrie moleciilaire des produits
organiques naturels. Soc. Chim. Paris, 1860. English translation in
Alembie Club Reprints, 14, 1860.
PEARSON, K., Nature, 58, 495 ; 59, 30, 124, 1898.
PRINGSHEIM, H., Z. physiol. Chem., 65, 89 and 96, 1910.
RITCHIE, P., Asymmetric Synthesis and Asymmetric Induction. Oxford,
1933.
ROSENTHALER, L., Biochem. Z., 14, 238, 1908.
SCHULZE, E. and BOSSHARD, Z. physiol. Chem., 10, 134, 1886.
SPENCER, H., Nature, 58, 592, 1898.
SPIERS, C, Naturwiss., 25, 457, 1937.
STOLL and WIEDEMANN, Uelv. Chim. Acta, 16, 307, 1933.
STRONG, W. M., Nature, 59, 53, 1898.
TOMIYASU, Y., Biochem. Z., 292, 234, 1937.
WALKER, W\ and KRIEBLE, W., J. Chem. Soc, 95, 1437, 1909.
CHAPTER III
HEREDITY AND THE INFLUENCE OF ENVIRONMENTAL
FACTORS ON THE OPTICAL ACTIVITY OF
BIOLOGICAL MATERIAL
Pasteur (1860) wondered how living beings would dif-
fer from what they are if the basic chemical substances
which compose them would change the sign of their opti-
cal rotation. Emil Fischer (1890) attempted to show how
this question can be answered experimentally. ''If it
proves possible", he writes, '"to feed plants, moulds or
yeasts with unnatural optical isomers of different sub-
stances, should one not expect that such a change in the
constructive material would result in a modification of
the delicate molecular architecture and of the entire
structure of organisms ? The biologists have not yet
studied this question because the chemists have not given
them the substances necessary for such experiments". At
present, fifty j^ears after Emil Fischer made this state-
ment, we know that such transformations of biological
structures are impossible, as the following data will show.
1. The Impossibility of Iiirertinf/ the Optical Proper-
ties of the Primary Constituents of Protoplasm. It is now
well established that, if one gives to microorganisms
unnatural food material as, for instance, laevorotatory
leucine, they are simply unable to make use of such food
as they do not possess the suitable enzymatic outfit. This
was soon ascertained by Fischer himself who found that
laevorotatory glucose is practically not fermented by
yeast cells (Fischer and Thierf elder, 1894).
In the light of modern knowledge one would not expect
that a simple change in nutrient conditions would deter-
59
60 orr.M rn .. h i:in:niTy a\d I':\] ihoxmext
mine the sign of asymmetry of protoplasm, wliicli is,
according- to all evidence, the result of a long evolution-
ary development.
Among more recent investigations on the inefficacy of
culture media on the sign of optical activity of primary
constituents of protoplasm, we shall mention those of
Gause and Smaragdova (1938). These authors have
studied the etfect on the yeast, Torula iitilis, of a pro-
longed cultivation in the optical isomers of leucine. The
yeast was cultivated at 27° C on pure left leucine in one
series and on pure right leucine in another. During the
iy2 months that the experiment lasted, 14 passages were
made. At the end of this period the rate of growth in
both series was measured. A similar procedure was fol-
lowed for a study of the action of right and left valine.
It was found that, though growth of Torula proceeds
much more rapidly on the biological forms of these amino-
acids (laevorotatory leucine and dextrorotatory valine),
there is some growth on their optical antipodes. It should
be noticed that the majority of other yeasts cannot grow
at all on the unnatural isomers of amino-acids. The very
possibility of a weak but unlimited growth of Torula
utUis on the right leucine and on the left valine is corre-
lated with the fact that these substances are first deamin-
ated by yeast (leucine, according to Ehrlich, 1906, is
transformed into iso-amyl alcohol), and the ammonia
thus formed proves to be a sufficient source of nitrogen
for the unlimited growth in Torula utilis. But the supply
of nitrogen in the form of ammonia alone is not sufficient
for most of the otljer species of yeast and a prolonged
growth on the unnatural isomers of amino-acids cannot
take place in them.
As was to be expected, a prolonged cultivation of Torula
on such nonbiological isomers had no essential influence
on the asymmetric properties of the protoplasm of these
yeasts, so that growth always remained more rapid on the
natural amino acids. Structurally inappropriate isomers
orr. ACT I v.. in:in:niTY \\i) r:\]Jh'o\]iE\T 61
cannot be directly involved in nietabolisiii. I'lie slight
growth observed on aniino-acids which can be deaminated
and thus transformed into material deprived of dissym-
metry, from which subsequently molecules of suitable
properties are built, even confirms this view. This con-
clusion is in accord with a number of recent observations
(Conrad and Berg, 1937; Du Vigneaud et al., 1939; Rat-
ner, Schoenheimer and Eittenberg, 1940).
2. The ImpossihUity of Modifying Protoplasm so as
to Cause it to Invert the Optical Properties of the Prod-
ucts of its Metabolism. That some products of meta-
bolism, that is, some secondary constituents of living mat-
ter may be generated in either of the two optically
isomeric forms, under the influence of external conditions,
has been admitted for a long time by a number of inves-
tigators. The case of lactic acid fermentation by some
microbes is classical in this respect. Concerning this
case we shall mention first the fundamental facts on the
specificity of each bacterial strain in the production
of one type of lactic acid and then we shall review the
more important investigations on the effect of external
conditions on such production.
Nencki (1891) showed that the optical form of the lac-
tic acid i^roduced in microbial fermentation is specific
for the kind and strain of microbes. Some species pro-
duce the pure dextrorotatory isomer, others give the
pure laevorotatory one and still others produce a form
of lactic acid which is either totally or partially racemic.
The sign of the asymmetry of the secondary substances
seems, therefore, to represent a stable hereditary char-
acteristic of the physiological organization of the cell
which has produced these substances and Nencki even
proposed to employ it for the identification of bacteria.
These observations were subsequently confirmed by Cur-
rie (1911), Pederson, Peterson and Fred (1926) and by
Katagiri and Kitahara (1937).
62 OPT. A CTIY., HEREDITY AND ENVIRONMENT
As to the problem of the possibility that different
isomers of lactic acid be produced when conditions of
cultivation are changed, the data of the literature have
for a long time been somewhat contradictory.
a. I n f 1 u e n c e of General Culture Con-
ditions. It was Pere (1893) who claimed for the first
time that one and the same line of Bacterium coli^ in dif-
ferent culture conditions can produce the two inverse
forms of lactic acid (cf. also Pottevin, 1898, and Pere,
1898). But to what extent the strains of microbes used
by them were bacteriologically pure is not clear.
Pederson, Peterson and Fred (1926) showed that in
mixed cultures of microbes consisting of producers of
the right and left isomers of lactic acid, one can often
observe a change in the form of the lactic acid produced
when the temperature, for example, is changed. How-
ever, in such cases, there is evidently no inverting mech-
anism in protoplasm. The species and strains of mi-
crobes which produce the right and the left isomers of
lactic acid possess different temperature optima of
growth and metabolism and consequently, in some con-
ditions of cultivation, the strains which produce the right
isomer and, in other conditions, those which give the left
isomer of lactic acid predominate. With microbic mate-
rial of a guaranteed purity having originated from one
cell, the possibility of inverting the lactic acid produced
has not been observed even in the most dift'erent culture
conditions, though the only isomer produced may subse-
quently undergo racemisation to various degrees.
6. I n f 1 u e n c e of the Culture M e d i u m.
In the literature the work of Kayser (1894) is often re-
ferred to as confirming Pere 's data, whilst, as a matter of
lit is to be pointed out that Bacterium coli does not cause a pure
lactic acid fermentation. Approximately half of the sugar is fermented
into lactic acid, the remainder is transformed into acetic acid, ethyl
alcohol, carbonic acid and hydrogen (Neuberg and Gorr, 1925). After
this was shown, the investigators began to work with true lactic-acid-
producers such as Lactobacillus.
Orr. ACT IV.. HFJihUUTY A^'D ENVIRONMEl^T 63
fact, the results of these two authors do not agree. Kay-
ser studied the formation of the optical isomers of lactic
acid by different strains of microbes cultured on various
sugars. He used 14 bacterial species or strains, with
which he performed 61 experiments. His data, which are
sunmiarized in Table 10, show that there are strains which
always produce the laevorotatory lactic acid, others
which always produce the dextrorotatory isomer and
finally some which yield a racemic mixture. Kayser's
results (1894), therefore, coincide not with Pere's (1893)
but with the data of recent investigators, particularly
with the thorough observations of Katagiri and Kitahara
(1937). In only one of the 61 experiments of Kayser (the
last one marked w^ith an asterisk in the table) was dextro-
rotatory lactic acid produced on one sugar (glucose) and
laevorotatory lactic acid on another (maltose). Since
this single excej^tion might have resulted from an in-
sufficient purity of the bacterial culture employed (cf.
Pederson and his collaborators, 1926), one comes to the
TABLE 10
ixfluexce of various sugars i.\ the nutritive medium ox the optical
Properties of the Lactic Acid Produced by Different Bacteria
(Kayser, 1894)
(The letters a, b, c, . . . refer to the species and strauis of bacteria,
most of which were not completely identified; the letters 1, d. and dl
indicate the optical rotation of the lactic acid produced; the figures
(1) and (2) after Maltose and Lactose mean that two media of dif-
ferent composition were used with each one of these sugars.)
Sugar
a
b
r
c
d
e
s
g
h
1
m
n
0
P
Arabinose
1
Xylose
1
Mannite
1
Glucose
1
1
1
1
1
d
dl
1
d
d*
1
dl+1
1
1
Le\ailose
1
dl
1
Galactose
1
1
1
dl
Maltose (1)
1
1
1
1
dl
d
1
dl
dl
dl
dl
Maltose (2)
1
Lactose (1)
1
1
1
d
1
d
dl
1
1
Lactose (2)
dl
1
1
Sucrose
1
d
dl
d
1
1
dl
dl
Melezitose
1
1
Trehalose
1
Starch
1
1
64 OPT. A CTn ., HEREDITY A^D EX 1 IROXMEXT
conclusion that Pere's results are by no means confirmed
by Kayser's.
This same table shows that racemization is greatly in-
fluenced by the culture medium. A given strain of bac-
teria, when cultured on a definite sugar, forms an almost
optically pure lactic acid (Pederson and collaborators,
1926, had shown that it is never entirely optically pure),
while on another sugar, it forms a racemic mixture. This
is the case, for instance, in the strains g, m, n, o, p. Ped-
erson and his collaborators (1926) made further observa-
tions on this point.
There are also the recent researches of Tatum and his
coworkers (1932) in which 4 strains of lactic acid bacte-
ria producing laevorotatory acid, 3 strains producing the
dextrorotatory acid and 13 different strains of Clostri-
dium acetohutylicwn were used. These authors found
that lactic acid bacteria produce the optically pure form
of lactic acid when grown separateh/ and the racemic
form when grown in association with the microbe caus-
ing acetonebutylic fermentation {Clostridium acetohuty-
licum ) .
At first Tatum (1932) interpreted his results in the
light of the hypothesis of Orla- Jensen (1919) according
to which there are in the bacterial cell two independent
enzymes, one of which produced the right and the other
the left lactic acid. In consequence of the association
of lactic acid bacteria with the butylic bacteria the meta-
bolism of the former would change in such a manner that
both optical isomers of lactic acid would start to be pro-
duced. However, in his later work (1936), Tatum showed
that, in the association of the two types of bacteria, lactic
acid is always initially formed m the optically active
state by the lactic acid bacteria, and that Clostridium is
only responsible for the subsequent racemization. (It is
interesting to note that racemization takes place in the
presence of antiseptics, therefore it is of enzymatic na-
ture.) The investigations were continued by Katagiri
OPT. . 1 r '77 1 .. // i:ri:dit\ amj i:\ virosmext 65
and Kitahai-a [VXu) who eslablislicd that the lactic-acid
bacteria which produced racemic acid are also capable
of racemiziiig a ready-made active acid. This is per-
formed by a special enzyme, called by these authors race-
miase. Those species and strains of bacteria which, in
fermentation, form as a tinal product optically active lac-
tic acid cannot racemize a ready-made active acid, they
have no racemiase. The formation of racemiase is closely
dependent on the culture conditions.
The results of the experiments of Katagiri and Kita-
hara (1937) are represented in Table 11. They show
clearly that, with various strains of Leuconostoc mesen-
teroides and Lactobacillus sake, a change of the condi-
tions of cultivation never brings about a change in the
type of the lactic acid formed ; a greater or lesser degree
of racemization of the substance produced accounts for
all the observed facts.
c. I n f 1 u e n c e of T e m p e r a t u r e. Similar
results were reported also in investigations on the in-
TABLE 11
Inflxjence of the Sugar Fermented ox the Optical Foi:m of the
Lactic Acid Produced in the Fermentation
(Katagiri and Kitahara, 1937)
(The letters 1, d and dl indicate the optical rotation.)
Microorganism Glucose ' Fructose
Mannose Galactose Arabinose Xylose
Leuconostoc
No. 34
1
1
1
—
1
1
No. 52
1
1
—
1
1
1
No. 13
1
1
—
1
—
—
No. 14
1
1
—
1
—
—
Lactobacillus
No. 41
d
—
d
d
—
—
' No. 24
d
d
—
—
d
—
' No. 53
d
d
—
—
d
—
No. 37
dl
dl
—
—
d
—
' No. 42
dl
dl
—
—
d
—
No. 45
dl+d
—
dl+d
dl+d
d
—
No. 57
dl+d
—
—
—
d
—
" No. 58
dl + d
—
—
—
d
—
66 OPT. ACTIV., HEREDITY AXD EXVTRONMENT
fiuence of nitrogen nutrition and of temperature upon the
optical form of the lactic acid obtained in bacterial fer-
mentation. The effects of various incubation tempera-
tures are presented in Table 12. One sees that the tem-
perature does not influence the sign of the optical rotation,
but that the degree of racemization of the acid regularly
increases with the rise of temperature.
Thus the old data of Kayser (1894) become clear. In
ditferent conditions of culture, the lactic acid formed by
a specific strain of bacteria possesses a different degree
of optical purity depending on the quantity of racemiase
contained in the bacterial cells.
Recently Kopeloff (1937) has shown that racemiase is
sometimes lost in the transition of the R-f orms of lactic
acid bacteria into the S-forms.
To summarize, the production of a specific optical
isomer, in the case of such secondary substances of the
protoplasm, or products of metabolism, as lactic acid,
represents a fixed hereditary character which is not de-
pendent on the conditions of cultivation. It is only such
processes as the velocity of a catalytic racemization of
secondary substances initially formed in the optically
pure state and the formation of racemiase which are de-
pendent on the culture conditions. The hereditary char-
TABLE 12
Influexce of the Temperature of Incubation upon the Optical Form
OF the Lactic Acid Obtained in Fermentation
(Katagiri and Kitahara, 1937)
(The letters 1, d and dl indicate the optical rotation.)
Microorganism
Temperature
30OC
20°C
6°C
Leuconostoc No. 14
1
—
1
No. 6
1
1
—
Lactodacillus No. 41
d
—
d
No. 42
dl
—
dl + d (17%)
No. 24
dl+d (39%)
dl+d (59%)
—
No. 45
dl-l-d (10%)
dl+d (47%)
orr. A(:ti\ .. // hredity asd environment 67
acter of optical activity in secondary constituents of pro-
toplasm and its independency on the external conditions
indicates that some physiological mutations peculiar to
some specific strains of bacteria must have occurred
some time in the past in those of them which produce opti-
cally unusual isomers.
Let us now attempt to penetrate into the nature of the
process by which a given isomer arises instead of its
antipode. This problem is directly related with the study
of some of the basic principles which underly the forma-
tion of physiological mutations.
3. Mechanisms Controlling the Production of a Given
Optical Isomer.
a. Production of Dissymmetric S u b-
stances from Symmetric P h e n y 1 - G 1 y o x a 1.
The observations of various authors concerning the trans-
formations of phenyl-giyoxal, a substance deprived of
dissjmimetry, into mandelic acid which possesses an asym-
metric carbon atom, are important in the study of the
question here discussed. These transformations are cat-
alysed by enzymes known generally as ketonaldehydemu-
tases. Starting from a symmetric initial product these
enzymes synthesize directly, without any intermediate
racemic stage, the optically active mandelic acid. Fur-
thermore, ketonaldehydemutases of ditferent species of
bacteria synthesize from the same initial product sub-
stances which are optically inverse, as the results re-
ported by different authors and represented in Table 13
show.
The action of the ketonaldehydenmtases is probably to
be attributed to the asymmetric state of these enzymes.
There are many observations more or less directly re-
lated to those just given. Neuberg and Simon (1926), for
example, found that an acetic-acid bacterium, B. ascendes,
produced laevorotatory amyl alcohol from a racemic
valeric aldehvde, while another bacterium of acetic acid
68 OFT. ACTI v.. HEREDITY AND ENTIF0X3IEXT
fermentation, B. pasteurianuni, formed dextrorotatory
amyl alcohol (an excess of 5 to 18 per cent) from the same
initial aldehyde. Analogous results were also obtained
with Bacterium pasteurianum in acetone preparations.
6, P r o d u c t i 0 n of Optical Isomers by
Esterases. The data of Willstatter and his collabor-
ators on the stereochemical specificity of esterases, the en-
zymes which catalyse the hydrolysis of the ethers of dif-
ferent organic acids, are of special interest in the present
problem. Some of these data are presented in Table 14,
but for more complete information we refer the readers to
the review by Rona and Amnion (1933).
In the majority of cases the esterase from liver and
the esterase from pancreas catalyze the hydrolysis of op-
tically inverse forms in initial racemic substrates.
c. P r o d u c t i o n of Optical Isomers by
Optically Active Alkaloid Catalysts.
Bredig and Fajans (1908) and Fajans (1910), in their
classical investigations on the decomposition of racemic
TABLE 13
Optical Activity of Maxdelic Acid Produced by Various
Microorganisms from Phenyl-Glyoxal
Organism
Initial
Substance
Mandelic Acid
Obtained
Author
1. Bacterium ascendes
Phenyl-glyoxal
d{-)
about 100%
Mayer, 1926
2. Lactobacillus 48
((
1( + )
84%
t(
3. The same; Acetone
((
1( + )
ft
preparation
4. B. (lelbruccki
(<
1( + )
82%
Neuberg and
Simon, 1927
5. B. lactis aerogenes
f (
d(-)
10%
ti
6. B. proteus
7. The same; Acetone
preparation
Phenyl-glyoxal
hydrate
it
d(-)
d(-)
95%
78%
Hayashi, 1929
t(
8. B. fluorescens
((
d(-)
43%
ti
9. B. jyyocyaneum
a
d(-)
37%
it
10. B. procligiosum
it
d(-)
87%
ti
11. B. coli
ti
d(-)
68 to 75%
tt
12. Parts of green
plants
Phenyl-glyoxal
d(-)
about 100%
Binder-
Kotrba, 1926
OPT. ACTI \ ., HEREDITY AND ENVIRONMENT 69
cam]ilioro-cai'boiiic acid into caini)lior aiul carbonic acid
under the inHiience of catalysts (optically active alka-
loids), have established that laevorotatory quinine cata-
lyses a more rapid decomposition of the left camphoro-
carbonic acid, while dextrorotatory quinidine causes a
more rapid decomposition of the right camphoro-car-
bonic acid.
TABLE 14
Sign of the Optical Rotation of the Component which is more Rap-
inLY HYPitni.YSED IX A Race:\iic Ether rxDER the Action of
Esterases of Different Origin
(CF. Rona and Ammon, 1933)
Initial i-acemic substrate
Esterase from
Pig's Pancreas
Esterase from
Pig's Liver
Mandelic acid — methyl ether
" " ethyl ether
" " monoglyceride
Phenylmethoxyacetic acid — methyl ether
Phenylchloroacetic acid — methyl ether
Phenylaminoacetic acid — propyl ether
Tropic acid — methyl ether
(-)
(-)
(-)
(-)
(-)
( + )
( + )
( + )
( + )
( + )
( + )
(-)
( + )
(-)
A similar condition has been observed in the synthesis
of optically active substances from structurally inactive
material. Bredig- and his collaborators, in their study of
the synthesis of nitrites from hydrocyanic acid and dif-
ferent aldehydes with the aid of optically inverse cata-
lysts of known chemical constitution, have obtained the
results reported in Table 15.
So the optically inverse catalysts, quinine and quini-
dine, bring about the synthesis of optical antipodes of the
nitrite of mandelic acid from initial structurally inactive
material. Quinine behaves in this respect analogous to
emulsin, while quinidine has the properties of the anti-
pode of emulsin. If the optically active catalyst, there-
fore, is of a given pign, it affects in a definite direction
the products of the catalyzed reaction.
''/. I^ r () d u c t i o n of a G i v e n 0 p t i c a 1
Isomer by a Chemical Alteration of
the Catalyst. In the cases studied in the preced-
70 O FT. A CTIY., HEREDITY AND ENVIRONMENT
ing pages substances of different optical signs result from
the action of optically active catalysts of different signs.
One of the two inverse catalysts might have originated by
an inverson from the other, but another possibility is that
the original catalyst has been changed chemicall}^ so as
to produce an optically inverse substance without being
itself inverted. The following case illustrates this last pos-
sibility. In the study of the enzymatic hydrolysis of
racemic ethyl mandelate by the esterase of the human
liver, it has been found that if one adds some strych-
nine, one obtains a strongly laevorotatory mandelic acid
instead of the usual dextrorotatory one (Bamann and
Laeverenz, 1930). Strychnine does not influence the vel-
ocity of hydrolysis of the right ether, but it strongly in-
creases that of the left ether thus causing the formation
of an excess of laevorotatory material (for further de-
tails see Rona and Amnion, 1933). There was no optical
inversion of the enzyme but the chemical properties of
the latter have been changed by combination with strych-
nine.
e. C o n t r o 1 of the Production of Op-
tical Isomers by Intermediate * ' P a t h-
w ays ". Let us now consider in greater detail the case
TABLE 15
Optical Pkoperties of the Nitriles Synthesized from Hydrocyanic
A( ID AND VAiiioiw Aldehydes undei: the Action of Different
Organic Catalysts
Initial aldehyde
Catalysts
Synthetic nitrile
Author
Benzaldehyde
Emulsin
d — nitrile
Rosenthaler,
1908
((
Quinine
d — nitrile
Bredig and
Fiske, 1912
a
Quinidine
1 — nitrile
**
Cinnamic aldehyde
Emulsin
d — nitrile
Rosenthaler,
1909
(( ((
Quinine
d — nitrile
Bredig and
Minaeff, 1932
a «
Quinidine
1 — nitrile
a
OPT. ACTIY., HEREDITY AXD ENVIRONMENT 71
so much investigated of the production of one optical
form of hictic acid by one kind of organism and of the
production of the other isomer of lactic acid by other
organisms.
An important contribution to the study of this phe-
nomenon has been brought forward by Embden, Baldes
and Schmitz (1912) who discovered that in the transfor-
mation of glucose into lactic acid by different animal tis-
sues dextrorotatory lactic acid is produced exclusively.
Another important advance was the finding of Neuberg
(1913) that extracts of animal tissues transform ethyl-
glyoxal, a structurally inactive body, into laevorotatory
lactic acid. A number of papers were then published on
the methyl-glyoxal reactions. It was found that in all
cases when methyl-glyoxal is converted into optically ac-
tive lactic acid the latter is laevorotatory. This was ob-
served, in particular by Neuberg and Kobel (1927) with
the yeast, Sacfiharomyces cerevisiae, by Neuberg and
Simon (1928) with Mucor javanicus and by Widmann
(1929) with Bacterium fluorescens.
From these observations Embden, Deuticke and Kraft
(1933) drew the important conclusion that since in tis-
sues of higher animals pure dextrorotatory lactic acid is
always formed and since the same tissues transform
methyl-glyoxal into laevorotatory lactic acid, methyl-
glyoxal cannot be the precursor of the dextrorotatory
lactic acid which appears in normal metabolism. Embden
then developed his theory of glycolysis in muscle which
received general acknowledgment. But, so far as we are
concerned in the present review, the essential fact is that
both optical isomers of a certain substance can appear in
metabolism when different intermediate substances are
involved. The left isomer of lactic acid is obtained
from glucose if the intermediate is methyl-glyoxal, and
the right isomer of lactic acid if the intermediate is,
according to current views, pyruvic acid. Embden sug-
gested that one or the other of these ^'pathways" could
Z^--
'JU.( / ,
72 OPT. AVTl 1 ., HEREDITY AM) EX 1 Ih'OXMEXT
be followed in the cells of different organisms and then
the production of the right or the left isomer of lactic
acid by various types of bacteria or tissues would be
accounted for. A physiological mutation which brings
about an optical inversion of the secondary protoplasmic
constituents may consequently consist in a change of the
intermediate pathways in the transformation of these
substances.
It appears then that the right and the left form of a
substance should not be considered so fundamentally op-
posed as far as their production is concerned, since one
has only to change the path followed in the transforma-
tions to obtain one or the other.
What happens when a right or left isomer originates
might also happen when the relative configuration of
optically active organic substances is concerned. In the
majority of cases optical isomers entering into the com-
position of living systems possess the same relative con-
figuration. Thus the configuration of natural alanine is
the same as that of natural ephedrine (Freudenberg and
Nikolai, 1934) ; the configuration of natural proline is the
same as that of natural nicotine according to Karrer
(see Pfeilfer and Christeleit, 1937). But there are also
cases in which the substances which constitute living sys-
tems belong to different series. Thus dextrorotatory lac^
tic acid, which is so generally found in the tissues of
higher animals, possesses the same relative configuration
as the unnatural laevorotatory tartaric acid, which is
never found in organic material. (It also has the same
configuration as natural dextrorotatory alanine; cf.
Freudenberg and Rhino, 1924, and Freudenberg, Brauns
and Siegel, 1923). The unity or the diversity of the rel-
ative configuration of organic substances, as well as the
character of being dextro- or laevorotatory, might depend
onl}^ on the biochemical path followed in the formation
of the substance.
OI'T. A(TI\ .. H Eh'HniTY WD i:S \ I ROX M EXT 73
/. V o 11 t r () 1 of the I * r o d u c t i o n o t' U p -
t i c a 1 isomers by an Inversion of
t li e W a 1 d e n Type. "Walden (1905) has shown
that some optically active substances, when subjected to
a series of substitution reactions, come out inverted. For
example, dextrorotatory alanine treated w^ith bromides
forms l-bromopropionic acid and, upon reaction with am-
monia, alanine is again obtained, but laevorotatory alan-
ine. Such a process is known as the ''Walden inver-
sion" and, according to Eniil Fischer, it is "the most
remarkable finding in the field of optical activity since the
fundamental investigations of Pasteur".
The mechanism of this inversion is far from clear.
In reactions of a certain type the asymmetric carbon
atom must be acted upon in such a manner that the con-
figuration of the molecule is inverted. In the example
given above one cannot even say if this is accomplished in
the transformation of alanine into bromopropionic acid
or in the transformation of bromopropionic acid into
alanine.
Mills (1932) had attempted to explain the Walden in-
version on the basis of some peculiarities of substitution
reactions. According to Levene, Rotheii and Kuna (1937),
there does not seem to be any general agreement on this
point.
Some have assumed that in biochemical reactions the
presence of a special enzyme, the waldenase, would be
responsible for the Walden inversion of some amino-
acids. This assumption, however, does not seem to stand
a critical stud3\
Walden inversion might perhaps play a role in some
biochemical processes, such as in the formation by some
bacteria of laevorotatory lactic acid from dextrorota-
tory glucose (see Freudenberg, Brauns and Siegel, 1923),
while other bacteria form dextrorotatory lactic acid. This
assumption, however, is not in the trend of current bio-
chemical theories. It is generally .supposed that, in the
74 OPT. A CTIV., HEREDITY AND ENVIRONMENT
formation of the left lactic acid, the structurally inactive
methyl-glyoxal represents an intermediate stage and,
according to Embden's scheme, in the formation of dex-
trorotatory lactic acid, the structurally inactive pyruvic
acid is the intermediate stage. The dissymmetric con-
figuration of the molecules is believed to disappear in the
intermediate stages of transformation and then to re-
appear again. In this interpretation one assumes that
the asymmetry of molecular aggregates has disappeared
in the intermediate stages because of the loss of dis-
symmetry of the molecules.
However, it is not necessarily so and Neuberg (1913),
questioning such intermediate loss of dissymmetry,
brought forward the theory of ''temporary dissymmetric
substances." If, for instance, in the intermediate stages
of transformation, methyl-glyoxal possesses some H and
OH groups attached to it, the dissymmetric configuration
of molecules will not disappear nor the asymmetric struc-
ture of molecular aggregates; then it would be possible
that in the production of one of the two optical isomers
of lactic acid from an initial active glucose by one type of
microbes a Walden inversion of the configuration of mole-
cules takes place.
The various questions studied in the last two sections
suggest the two following generalizations which may be
of significance in understanding the basic principles of
vital activity : 1 . The impossibility of altering the optical
properties of the primary substances of protoplasm fits in
with the assumption that the activity of the fundamental
physiological systems is based upon the principles of
''fixed pathway", i. e., all the intermediate transforma-
tions in these physiological systems would proceed along
definitely fixed paths. On the contrary, the optical prop-
erties of the secondary protoplasmic substances can be
altered to a certain extent. Consequently, the formation
and the transformations of the secondary substances are
not bound by the principles of fixed pathway. 2. Fur-
OPT. ACT IT., HEREDITY AND ENVIRONMENT 75
thermore, in the fundamental protoplasmic systems, there
are devices to avoid racemization, which were discussed
previously; similarly it seems that there are devices to
avoid inversion, such as those regulated by the ''prin-
ciple of fixed pathway". Neither of these devices oper-
ates in the transformations of secondary protoplasmic
constituents.
SUMMARY
1. It is not possible to invert, by external influences,
the asymmetric structure of the primary constituents of
protoplasm which is the result of a long evolutionary
process.
2. As to secondary substances of protoplasm such as
products of metabolism (for example, lactic acid in fer-
mentative processes), the sign of their optical activity
also represents a fixed hereditary character of the spe-
cies or strain which elaborated them, but external in-
fluences can affect the catalytic racemization of these
products which were initially formed in the optically pure
state.
3. Concerning the mechanism by which the produc-
tion of a given optical isomer is controlled in metabolic
activities, one should note that: (a) From the same
symmetric initial substrate, enzymes of ditferent organ-
isms can synthesize optically dilferent substances; (b) It
is often through optically inverse catalysts that optical
antipods are synthesized; (c) Some catalysts can, after
chemical alterations which do not constitute an inver-
sion, synthesize substances in a form which is the optical
inverse of the form that was synthesized before the al-
teration of the catalyst; ( d) The sign of the optical rota-
tion of the final product of a series of metabolic reactions
may depend on the "pathway" followed in intermediate
reactions; (e) Inversions of the Walden type (transfor-
mation of one optical isomer into its antipod in a series
76 OPT. A CTI I . , HEREDITY AND EN VI RON M EN T
of chemical transformations) may play a role in biologi-
cal phenomena.
4. Physiological mutations which, in the evolutionary
development of an organism, bring about optical inver-
sions of the secondary protoplasmic constituents may con-
sist in a change of the intermediate "pathways" in a
series of reactions.
5. Protoplasmic systems are provided wdth mechanisms
by which "pathways" that would lead to an inversion of
the asymmetric structure are avoided. Primary constit-
uents of protoplasm are then regulated by a principle
called here: ''Principle of fixed pathway". Such mech-
anisms do not operate in the transformations of the sec-
ondary substances of protoplasm.
BIBLIOGRAPHY
BAMANN. E. and LAEVERENZ, P., Ber. vhem. Ges., 63, 394, 1930.
BREDIG, G. and FAJANS, K., Ber. chem. Ges., .',1. 752, 1908.
CONRAD. R. and BERG, C J. Biol. Chem., Ill, 351, 1937.
CURRIE. J. N., Bioch. Bull., 1, 103, 191.
DU VIGNEAUD, V., COHN M., BROWN, G., IRISH, O., SCHOEN-
HEIMER. R. and RITTENBERG, D., J. Biol. Vhem., 131, 273, 1939.
EHRLICH, F., Biochem. Z.. 1, 8, 1906.
EMBDEN. G.. BALDES, K. and SCHMITZ, E., Biochem.. Z.. J,5, 108,
1912.
EMBDEN, G.. DEUTICKE, H. and KRAFT, G., Klin. Wochenschr., 12,
213, 1933.
FAJANS, K., Z. physikal. Chem., 73, 25, 1910.
FISCHER, E., Ber. chem,. Ges., 23, 370, 1890.
FISCHER. E. and THIERFELDER, H., Ber. chem. Ges., 21, 2031, 1894.
FREUDENBERG. K.. BRAUNS, F. and SIEGEL, K., Ber. chem. Ges.,
56, 193, 1923.
FREUDENBERG, K. and NIKOLAI, F., Lieb. Ann. Chem., 510, 223,
1934.
FREUDENBERG, K. and RHINO, F., Ber. chem. Ges., 51, 1547, 1924.
GAUSE, G. F. and SMARAGDOVA, N. P., Biol. J. (Russian), 1, 399,
1938.
KATAGIRI, H. and KITAHARA, K., Biochem. J., 31, 909, 1937.
KAYSER, E., Ann. hist. Pasteur, 8, 737, 1894.
KOPELOFF, L. and N., J. Bad., 33, 331, 1937.
LEVENE, P., ROTHEN, A. and KUNA, M., J. Biol. Chem., 120, 111,
1937.
MILLS, W. H., J. Soc. Chem. Ind., 51, 750, 1932.
NENCKI, M., Zhl. Bakt., 9, 304, 1891.
OPT, ACTI 1 .. H EUEDl TY A\JJ ENVIRONMENT 77
NEUBERG, C, Biochem. Z., 51, 484, 1913.
NEUBERG, C. and GORR, G., Biochem. Z., KUl 482, 1925.
NEUBERG, C. and KOBEL, M., Biochem. Z., 182, 470, 1927.
NEUBERG, C. and SIMON, E., Biochem. Z., 179, 443, 1926; 200, 468,
1928.
ORLA-JENSEN. The Lactic Acid Bacteria. Copenhagen. 1919.
PASTEUR, L., Recherches sur la dissymetrie moleculaire des produits
oiganiques natux-els. Soc. Chim. Paris, 1860. English translation
in Alembie Club Reprints, 14, 1897.
PEDERSON, C, PETERSON, W. and FRED, E., J. Biol. Chem., 68,
151, 1926.
PeRe, a., A)iii. Inst. Pasteur, 7, 737, 1893; 12, 63, 1898.
PFEIFFER, P. and CHRISTELEIT, W., Z. physiol. Chem.. 2 ',.7. 197,
1937.
POTTEVIN, A., Ann. Inst. Pasteur, 12, 49. 1898.
RATNER, S.. SCHOENHBIMER. R. and RITTENBERG, D., .7. Biol.
Chem., 13-',, 653, 1940.
RONA, P. and AMMON, R., Ergehn. Ensymforsch., 2, 50, 1933.
TATUM. E.. PETERSON, W. and FRED, E., Biochem. J., 26, 846, 1932;
.361, 1892. 1936.
WALDEN, P., Ber. chem. Ges., 38, 345, 1905.
WEBSTER, M. and BERNHEIM, F., J. Biol. Chem., IIJ,, 265, 1936.
WIDMANN, E., Biochem. Z., 216, 475, 1929.
CHAPTER IV
ON THE RELATION BETWEEN THE INVERSION OF
SPIRALLY TWISTED ORGANISMS AND THE
MOLECULAR INVERSION OF THEIR
PROTOPLASMIC CONSTITUENTS
1. Morphological Dissymmetry and Morphological In-
version. The attention of biologists has for a long time
been attracted by the existence of dextral and sinistral
spirally twisted forms in some animal or plant popula-
tions. Ludwig (1932, 1936) published two extensive re-
views in which he summarized a large number of scattered
observations on this subject. These reviews show that
practically all the studies of dextrality and sinistrality in
plants and animals consist in descriptions of the morpho-
logical aspects of the phenomenon and that the physio-
logical mechanism which underlies the morphological
processes has been left almost untouched.
One of the basic attributes of spiral structures is their
ability to undergo genotypic inversion. The work of
Boycott, Diver, Hardy and Turner (1929) on the heredity
of sinistrality in the mollusc Limnaea peregra has shown
that the usual twist of the coil of this mollusc to the
right (clockwise) is determined by a dominant gene, while
the twist to the left is controlled by the recessive gene,
and that the sinistral mutant individuals appear in the
population from time to time. Consequently, in almost
all the cases in which some experimental work was car-
ried out with organisms possessing a spiral form, it
was possible to detect among the usual, typical individ-
uals a few hereditary inverse specimens. (We shall,
79
80 SPIRAL TMIST AXD OPT. AVTlYrVY
hereafter, call these two kinds the typical and the in-
verse individuals.)
It may be supposed that the direction of the spiral is
determined by some dissymmetric substance which is
labile, in the sense that it can undergo an inversion of
its molecular configuration with comparative ease, and
that by means of such a mutation, the form of the or-
ganism can change. Such an idea is due to Koltzoff
(1934) and to Needham (1934), according to whom the
origin of dextrality and sinistrality, as observed in the
eggs of certain snails and later in their body, is connected
with the stereo-chemical properties of some of their com-
ponent protein molecules. But, at present, these rela-
tions are very obscure.
Koltzoff expresses himself as follows : ''Particularly in-
teresting is the case when in a pond-snail, Limnaea ru-
hella, in one and the same species genotypes are found
which are characterized by either the left or the right
spiral types of shell. The cleavage of the ovum in those
genotypes proceeds, correspondingly, according to the
right or left spiral types. Here, already at the first
division of the ovum, the difference between both types
is marked in a sufficiently distinct manner by the posi-
tion of spindles. It is very probable that the right and
the left types are distinct already in the unfertilised
ovum, because they can be detected in the relative posi-
tion of both directing bodies. Hybridological analysis
shows that this character is determined by one pair of
allelomorphs. The right twist of the spiral is determined
by the dominant gene, the left twist by the recessive one.
The mother homozygous as to the recessive gene can
itself have the right spiral (because the ovocyte from
which it evolved could be heterozygous), but all its eggs
develop according to the left type, even if they were
fertilized by the sperm of the homozygous dextral fa-
ther. This shows that genotypical peculiarities of the
male nucleus are not manifest on this stage. On the
SPINA L TW Tf< T AND OPT. AdlVITY 8 1
other hand, one can see here the proof of the fact that
the basic features of the whole plan of structure of an
organism can be the result of the action of a single gene.
''What kind of influence this gene exercises on the
structure of the ovum, we certainly do not know. As a
hypothesis I can express the suggestion that this or
other type of cleavage is here determined by the presence
in the protoplasm of the ovum of the right or of the left
optical isomer of some organic substance. This sub-
stance goes out of the nucleus of the ovum during the
ripening of the latter, forming itself preliminarily in
connection with corresponding genes of the chromosome
apparate of the ovocyte. Hence it may be inferred that
both genes of a given couple of allelomorphs are optical
isomers in respect to each other. ' '
Koltzotf 's hypothesis is, of course, not the only possible
interpretation of the observed facts. The optical inver-
sion of genes is certainly possible, but a change of their
chemical properties, without the inversion of their con-
figuration, may also be supposed. In the latter case the
optical inversion of some organic substance determining
the structure of the animal would take place only in a
subsidiary reaction. The facts discussed in the preceding-
chapter would confer about the same degree of proba-
bility on the hypothesis of gene inversion and on the
hypothesis of a chemical modification of the genes with-
out inversion.
The solution of some basic biological problems de-
pends on the answer to this question. According to the
first interpretation, the substance of the genes which
determine the morphological structure of an animal would
belong to the group of secondary protoplasmic constit-
uents, those which play the role of storage substances or
of products of metabolism. These products would then be
quite important in the mechanism of evolution.
2. Morphological Dissymmetry luuJ Morphological In-
version in Bacillus Mycoides. The typical strain of Bacil-
82
SPIRAL TWL'^T A\D OPT. ACTIVITY
lus mycoides, when grown on the surface of agar pep-
tone medium, produces colonies spirally twisting to the
left, i. e., counter-clockwise (according to the terminology
adopted by Ludwig, 1932). After one has introduced a
small quantity of inoculating material in the centre of a
Petri dish of agar-peptone, one soon sees it grow ; the thin
filaments of the growing culture begin to deviate to the
left (cf. Fig. 5).
L
D
Fig. 5. Dextral (D) and sinistral (L) spiral twisting of the growing
filaments of colonies of Bacillus mycoides, as observed on peptone
agar, in Petri dishes.
The inverse form of this organism, growing in dextral
coils, rarely occurs. It was first recorded by Gersbach
(1922), who described this interesting case as an ''isom-
erism in bacteria". He further established that the
dextral and sinistral strains are entirely identical in all
their properties.
Later a single dextral strain among a great number of
sinistral ones was observed by Oesterle (1929).
Lewis (1932) isolated several dextral strains in Texas.
In an extended series of investigations with Bacillus
mycoides at the Microbiological Institute of the Academy
SPIRAL TWIST AND OPT. ACTHITY 83
of Sciences in Moscow, the dextral form was found only
three times, though numerous isolations from different
soils were made.
The dextralitj" and sinistrality in Bacillus mycoidcs is
a hereditar}^ feature. Dextral forms are always obtained
from dextral forms, and sinistral from sinistral ones.
It can be shown that the spiral form of the colonies of
this organism is a secondary feature which is the result
of the primary spiral structure of the growing cells which
constitute the filaments. If one stains the filaments on
the surface of the agar with neutral red or with toluidin
blue (1:5000) and examines them under the microscope,
one can occasionally observe the twisting of two filaments
which have encountered each other. The motion of the
growing filament consists of two components : an elonga-
tion and a rotation around the axis of the filament, these
will result in a spiral motion. Similar observations have
been made also by Stapp and Zycha (1931) and by Eob-
erts (1938). If during the free growth of a filament on
the agar surface, the filament rotates around its longitu-
dinal axis counter-clockwise, the interaction of the firm
surface of the agar and of the growing filament will cause
the latter to follow a spiral path in a counter-clockwise
direction. Consequently, the secondary sinistral coil of
the growing colony of bacteria will arise as a result of the
primary sinistral spiral growth of the cells of the fila-
ment. This is confirmed by the fact that a certain con-
sistency of culture medium is necessary for the typical
spiral growth of colonies (Pringsheim and Langer, 1924;
Hastings and Sagen, 1933). The latter authors state that
on agar of usual strength the growth of Bacillus my-
coides spreads from the place of inoculation in the form
of coarse filaments which twist counter-clockwise, form-
ing a symmetrical pattern. On less consistent agar this
pattern does not appear or is diffuse.
3. Morphological Dissymmetry and Morphological In-
version in the Snail, Fruticicola lantzi. We shall consider
84 SPIRAL TWIST AXD OPT. ACTTTITY
next the morpliological dissymmetry of an animal which,
in the natural classification, stands far from the bacteria,
namely, the land snail, Fruticicola lantzi. In this animal
the typical individuals are dextrally spiralled as is the
case in the majority of species of snails. Numerous ob-
servations have led to the conclusion that, in snails, the
sinistrally twisted individuals are ecologically weaker
than the dextral forms. In joint occurrence of the two
forms the sinistral ones often disappear in a rather
short time. Zvetkov (1938) has recently shown, in a
study of the distribution of Fruticicola lantzi in Middle
Asia, that most of the populations consist of typical
forms, dextrally spiralled. Populations consisting al-
most exclusively of inverse, sinistrally spiralled forms
were found only in some districts separated from the re-
maining area by mountain barriers. Such isolated col-
onies of inverse forms in both bacteria and hermaphro-
ditic molluscs, it is thought, have originated from a sin-
gle inverted ancestor.
I-. Some Physiological Properties of the Dextral and
the Sinistral Strains of BaciUus mycoides. a. A c t i o n
of temperature. Recent experiments made by
Gause (1939) have shown that there is a ditference in the
action of temperature on the growth of the dextral and
on that of the sinistral strains of Bacillus mycoides.
Three series of experiments were performed as follows :
A small quantity of inoculating material was placed in the
center of the Petri dish, on agar, in the form of a circle
0.5 mm. in diameter. Twenty hours after growth had
started the diameter of the colony was about 6 mm. at
20° C and about 25 mm. at 32° C. Taking then the diam-
eter of colonies, either dextral or sinistral, growing at
20° C, as a unit, curves of growth in terms of temperature
were constructed. Such curves are represented in Figure
6. If the rate of growth of the usual sinistral strain is
normal, the inverted dextral strain presents the jdIic-
nomenon of ''heat injury."
SPIRAL TWIST A\D. OPT. ACTIVPTY
85
While the preceding experiment was made on the rough
form (usually designated as the R-forni) of bacteria, we
repeated it with the smooth form (S-form). By "disso-
20° 24° 28'
32"" 36°C
Fig. 6. Growth-temperature relation in dextral (D) and sinistral
(L) strains of Bacillus mycoides (R type) grown on solid medium.
The three sets of curves represent three series of experiments. The
size of the colonies grown at various temperatures is indicated in
terms of the size of the colonies grown at 20° as a unit. (From Gause,
1939.)
86
^^PIRAL 7'TT7>ST AND OPT. ACT I \ IT Y
ciation" botli dextral, DR, and sinistral, LR, strains of
Bacillus mycoides develop into smooth ones, which form,
on growing on solid medium, flat spherical colonies, LS
and DS (for literature see Arkwright, 1930). The study of
the action of temperature was made according to the pre-
vious plan. It was found that the phenomenon of heat
injury characteristic of the dextral strains was about as
marked in the S-f orms as in the R-f orms.
It was decided then to try the experiment with the
DS and LS strains in liquid medium. The rate of growth
was now determined by a bacterial count in a Thoma
chamber, under the microscope, forty hours after the
inoculation. The relation of growth to temperature is
represented in Figure 7. One sees that in the dextral
strain the characteristic heat injury appears in the range
of temperatures extending' from 24° to 28° C.
Similar results were thus obtained with the R-forms,
with the S-forms and on liquid as well as on solid culture
200
I 50
100 -
Fig. 7. Growth-temperature relation in dextral (DS) and sinistral
(LS) strains of Bacillus ^nycoides, grown on liquid medium. Abscissae:
Temperature in degrees C; Ordinates: Number of cells per 1/160 cc.
(From Gause, 1939.)
SPIRAL TWIST Ay D OPT. ACTIYTTV 87
media. There is definitely in the dextral form a special
sensitivity to heat injury at the temperatures indicated.
It is interesting to compare this characteristic weakness
of the inverted dextral form with the following property
pointed out by Le^\ds (1933) and observed again by Gause
(1939).
b. Enzymatic properties. Lewis, working
with dextral and sinistral strains of Bacillus mycoides,
reported a specific physiological difference between them.
It is known that this bacillus possesses the ability to
decompose glucose and saccharose and to acidify the me-
dium. On glucose the acid production is similar in sinis-
tral and in dextral strains but not on saccharose. Accord-
ing to Lewis the formation of acid on saccharose is rapid
with the left spiralled strain and it ceases with the right-
spiralled type.
Gause (1939) repeated these experiments using the S-
form of Bacillus mycoides. Tw^o per cent saccharose and
a little quantity of a weak (0.02 per cent) solution of
phenol red (according to Clark) were added to an agar-
peptone medium of usual composition. At pH 7.7, which
is the optimal hydrogen ion concentration for the gro^\i:h
of Bacillus mycoides, phenol red has an orange tint. The
culture w^as kept at 28° C. Nineteen hours after the be-
ginning of the experiment the color of the sinistral
strains differed very sharply from that of the dextral.
The former showed a rapid production of acid, the pH
of the colony being about 6.8. The dextral strains showed
no production of acid. The reaction of the colony was
alkaline, its pH being about 8.4. Lewis' results were
therefore confirmed with the S-form of the bacillus.
It might be worth mentioning the following detail.
While the dextral strains are, without exception, unable
to produce acid on sucrose, the sinistral strains were all
able to form acid except in two cases, one mentioned by
Lewis and another (doubtful) observed by Gause.
88
.SPIRAL TWIST AND OPT. ACTIVITY
Lewis' reaction might be considered as a fermentative
deficiency of the dextral strain.
(7. G r o w t h on two optical isomers. It
is of interest to know how an organism which presents a
morphologically dissymmetric structure (right or left
spiral) behaves when grown on a medium which is molec-
ularly diss^anmetric. The dextral and sinistral strains
of Bacillus mycoides were growm on the optical isomers
of arginine (Gause, 1939). The results are summarized
in Tables 16 and 17.
A safe conclusion that one can draw from these data is
that both the dextral and the sinistral strains of Bacillus
mycoides, in both the rough and the smooth forms, grow
TABLE 16
Growth of Sinistral (LR) and of Dextkal (DR) Forms of Bacillus
Mycoides on Optical Isomers of Arginine
Amino
Number of
Ratio of
growth on d-ar-
ginlne to
growth on
dl-arginine
Number of
Ratio of
growth on
d-arginine
acid
cells in LR
cells in DR
to growth
on dl-ar-
ginine
d-arginine
31.3 1
15.7 1
19.7 i
dl-arginine
1.99
12.4 f
1.59
d-arginine
28.9 )
21.2 I
dl-arginine
14. 6 i
1.98
13.2 \
1.61
TABLE 17
Growth of Smooth Sinistral (LS) anu of Smooth Dextral (DS)
Forms of Bacillus Mycoides on Optical Isomers of Arginine
Amino
acid
Number of
cells in LR
Ratio of
growth on d-ar-
ginine to
growth on
dl-arginine
Number of
cells in DR
Ratio of
growth on
d-arginine
to growth
on dl-ar-
ginine
d-arginine
dl-arginine
d-arginine
dl-arginine
40.1 (
31.9 1
43.5 /
23.3 )
1.26
1.87
66.8 [
31.0 t
45.5 )
33.0 \
2.15
1.38
^^rih'AL T\\ IHT AND OPT. ACTIVITY 89
better on iiahiral d-arginiiie than on the racemic dl-ar-
giuine. Therefore, the dextrally and the sinistrally twist-
ed organisms are alike in the optical properties of their
basic protoplasmic constituents, since nutritive sub-
stances of the natural configuration are more favorable
for both of them.
d. E e s p i r a t i o n. The oxidation of glucose by the
dextral and sinistral strains of Bacillus mycoides was
determined by the Warburg technique at three tempera-
tures: 22°, 25° and 28°C. As is characteristic of biolog-
ical processes generally, the velocity of respiration rose
exponentially with the rise of temperature, and practi-
cally at the same rate in the sinistral and in the dextral
strains of the bacillus. Consequently the phenomenon of
heat injury, which was characteristic of the growth of
the dextral strain, was not observed in respiration on
glucose.
The general conclusions to draw from these investiga-
tions is that the inverse, dextral strains of Bacillus my-
coides are weaker than the typical, sinistral ones. This
was observed in the rate of growth at 24° to 28° and in
the deficiency of an enzymatic action.
5. Some Physiological Properties of the Dextral and of
the Sinistral Strains of the Snail, Fniticicola lantzi.
Anabolic gain, I'esistance to starva-
tion, mortality rate. The results of the in-
vestigations on Bacillus mycoides are paralleled by the
data obtained with Fruticicola lantzi.
Gause and Smaragdova (1940) made a comparative
study, under well controlled laboratory conditions, of the
physiological behavior of the dextral and sinistral indi-
viduals of this snail. They investigated (1) the velocity
of anabolic assimilation as judged by the change in weight
w^hen the snails were fed for a long time on carrots; (2)
the velocity of the catabolic loss as determined by the
decrease in weight when the animals were kept in a moist
chamber without food; (3) the mortality rate in the sec-
ond group of experiments.
90
SPIRAL TWLST AND OPT. ACTIVITY
It was found that after having been fed for a prolonged
time on carrots tlie typical, dextrally twisted individuals
practically did not change their weight, while, under per-
fectly identical conditions, the sinistrally twisted snails
considerablv decreased in weight (cf. Figure 8).
320 -
300.-
260 -
240 -
220 -
7\
DEXTRAL
^ -"' " "P^^
■ -u
/ ,» -^
\. /^
1 1
t 1
P'""^""^
SINISTRAL
-
■ 1
T :
9
10
20
30
40
50
60
Fig. 8. Change in weight observed in the dextral and sinistral forms
of the snail Fruticicola lantzi when they were fed for a long time on
carrots. Abscissae: Time in days; Ordinates: Weight in milligrams.
(From Gause and Smaragdova, 1939.)
In the study of the behavior of Fruticicola in starvation
it was observed that the sinistral individuals lost weight
more rapidly and died off more quickly than the dextral
forms (cf. Figure 9).
The relative weakness of inverse left spiralled indi-
viduals is evident in the three series of experiments.
Similar results were obtained also in the study of the
loss of dry weight.
SPIRAL TWIST A\D OPT. ACTIVITY
91
uu7o
V
90%
'v
\^
X o
80%
5\
V^
"""^^
70%
-
I
•^
^>--f
e
10
Fig. 9. Average decrease in fresh weight in starving adult dextral
(D) and sinistral (S) forms of the snails FrvAicicola lantzi. Abscissae:
Time in days; Ordinates: Weight in % of the initial value. (From
Gause and Smaragdova, 1939.)
6. On the Relation hetiveen MorpJiological Inversion
and Molecular Inversion. Having considered the physio-
logical differences of the dextral and sinistral forms, we
shall now turn to the problem of the possible relation
between morphological and molecular inversions. Let us
at first note that the heat injury in the dextral strains of
Bacillus mycoides reminds one of the heat injury observed
when different lower organisms, such as yeast, were cul-
tured on unnatural isomers of amino acids (Gause and
Smaragdova, 1938). When the yeast Torula utilis was
grown on the natural isomer of leucine, which enters into
the composition of all living organisms, the velocity of
growth was that always observed in typical growth-tem-
perature curves, but when it was cultured on the unnat-
ural isomer of leucine, the increase in the velocity of
growth became always less and less with the rise of tem-
perature.
92
.Sl'Ux'AL T]\J,ST AM) OPT. ACTITITY
These experiments were rei:)eated with the optical
isomers of the following- amino acids : leucine, histidine,
phenyl-alanine and valine (Gause, 1939). The results
obtained are given in Figure 10. Typical heat injury at
temperatures extending from 18° to 28° may be observed
in the growth of Torula nfills on the unnatural isomers
of leucine and of histidine but not on those of valine and
phenyl-alanine.
80-
60
40
20
25
20
140 iQO 22° 26°
18° 22° 26°C
10
5
Fig. 10. Growth of the yeast Torula iitilis on optical isomers of
various amino acids, at different temperatures. Abscissae: Temperature
in degrees C; Ordinates: Increase in the number of cells in 40 hours.
(Prom Gause, 1939.)
s/'iir\L T\\ 1ST A\n. oi'T. Acrn ITY 93
Similar data were obtained also in the study of the
growth of the- mould Aspergillus niger on the optical
isomers of leucine and valine.
The same relation between lenii)erature and rate of
growth is thus observed in the unusual strain of Bacillus
mycoides growni on natural substrates and in the yeast
or fungi grown on unnatural substrates. In both cases,
there nuist be an inhibitive factor of growth. It may be
conjectured that in the case of yeast or fungi the un-
natural isomer of the amino acid dissolved in the cul-
ture medium surrounding the cells caused a retardation
of growth because its spatial contiguration did not coin-
cide with the spatial configuration of the basic constit-
uents of protoplasm. In the case of Bacillus mycoides
the retardation of growth in the unusual form would be
caused by the presence inside of the cells of the unnatural
optical isomer of some organic substance which partici-
pated in the determination of the morphology of the
cell.
7. Morpliological Inversion and the Theory of Spiral
Growth. The investigations on various physiological
properties of dextral and of sinistral strains in Bacillus
mycoides and in Fruticicola lantzi have brought out the
two following points : ( 1) In the optical properties of their
protoplasm these strains are alike. This follows from
their behavior towards optically isomeric nutritive sub-
stances. It has, furthermore, been confirmed by direct
observations made by Kiesel, Efimochkina and Rail
(1939), wdio isolated the same natural amino acids from
both dextral and sinistral strains of the snail Fruticicola
lantzi. (2) The inverted individuals of both bacteria and
snails are physiologically weaker than the typical ones.
These observations suggest that while in the typical in-
dividuals the organic substances which participate in the
determination of the twist might well have the same laevo-
rotatory configuration as the other constituents of proto-
plasm, in the inverted individuals some enzymatic dis-
turbance might have occurred.
94 SPIRAL TWIST A^W OFT. ACTIVITY
Castle (1936) has recently undertaken the study of the
mechanism of spiral growth in Phy corny ces. He claims
that the spiral structure of the growing cell wall is not
strictly predetermined but depends on the interaction of
forces which exert their action in the growth region. The
twist of the growing elastic elements of the wall may be
the result of their resistance to turgor. Castle construct-
ed a model to illustrate this process. If the elastic ele-
ments of the wall are distributed symmetrically, dextral
spirals ^\i\\ be obtained in 50 per cent of the cases and sin-
istral spirals in the other 50 per cent. As the left direc-
tion of the spirals is typical for Phycomyces, one must
assume a dissymmetric distribution of the elastic ele-
ments of the cell wall, which later, under the action of
turgor, lead to the formation of sinistral spirals.
As Castle himself points out, the mechanism of spiral
growth can be different in different organisms, and one
cannot directly transfer his explanation of the mechanism
of twisting to the bacteria, the more so since the cell wall
of the latter is thought to consist of some specific pro-
tein material closely related by its nature and origin to
cellular protoplasm (John-Brooks, 1930).
But one can assume that in the spiral growth of the cell
wall of a bacterium, as in that of Phycomyces, tw^o fac-
tors are involved: (1) Some pre-existing asymmetric
system (distribution of the elastic elements of the wall
in Phycomyces; optically active secondary protoplasmic
constituents in bacteria) ; (2) a system of forces directly
inducing the spiral twist (turgor in Castle's experi-
ments). The interaction of these two factors would bring
about the dextrality or sinistrality of the spiral growth
according to the following scheme :
Secondary substance Asymmetric structure Sniral growth
of metabolism ^ of the cell wall ^ ^
f
Forces directly inducing
* the spiral twist
SPIRAL TWTST AND OPT. ACllYTTY 95
One may conjecture that the inversion of the direction
of the spiral growth in Bacillus mycoides is related to an
optical inversion of some secondary substance in meta-
bolism. The latter would bring about the inversion of
some structures in the cell wall and, from the interaction
of these with the forces inducing the spiral twist, there
would result an inversion in the direction of the spiral
grow^th of the cells.
But to what extent can one assume that the secondary
substances of metabolism which participate in the struc-
ture of the wall of the bacterial cell can undergo an optical
inversion? Some recent data on the chemical structure
of bacterial capsules obtained by Bruckner and Ivanovics
(1937) in the laboratory of Professor Szent-Gyorgyi, are
of interest in this connection. As is kno^vn, the cell wall
in bacteria consists of two layers: (1) A very thin in-
ternal cuticle, and (2) An external gelatinous layer which
is sometimes developed into an envelope called a capsule.
(John-Brooks, 1930, remarks that the bacteriologists have
come to look upon capsule formation as a general feature
which is common to all bacteria, but which reaches the
proportions that we know, only in certain species.) Bruck-
ner and Ivanovics (1937) w^ho studied the chemical prop-
erties of the capsule of Bacillus antJiracis and of some
other species of bacteria, all of which are aerobic spore
formers, standing near Bacillus mycoides in the classifi-
cation, found that the capsule of these bacteria consists of
a polypeptide substance, the hydrolysis of which yields
d(-) glutamic acid. The laevorotatory isomer of this
amino acid is unnatural, and it has not been found pre-
viously anywhere in the organic nature. So, the presence
of the unnatural glutamic acid in the structure of the
envelope of the anthrax and of some other bacilli has
already been recorded in the literature. Further investi-
gations in that direction may reveal significant data on
the present problem.
96 S/'lh'AL TW'/sr A.\l> OI'T. ACTH IT)
SUMMARY
1. In organisms which possess a spiral structure, as in
some bacteria, in snails, etc., one observes a larger num-
ber of "typical" individuals, that is, of individuals twist-
ed in one direction, while the "inverse" specimens are
rarer.
2. The properties of protoplasm related to optical ac-
tivity are alike in dextral and sinistral forms. The same
natural amino acids have been isolated from either the
dextral or the sinistral snails {Fruticicola laiitzi). Both
dextral and sinistral bacteria {B. my cold es) grow better
on the natural than on the unnatural isomers of amino
acids.
3. The "inverse" forms are physiologically weaker
than the "typical." When the culture temperatures are
varied from 20" to 36% the "inverse" bacteria present a
decreasing growth rate not observed in "typical" bacte-
ria; furthermore, the "inverse" forms show some enzy-
matic deficiencies. In the "inverse" snails the velocity
of catabolic loss and the mortality rate, on starvation,
exceed those of the "typical" individuals.
4. It is suggested that some secondary substances which
may determine the morphological inversion are optically
inverted, or that some subsidiary process in metabolic
activities is changed in the nmtant snails and bacteria,
whereas the basic protoplasmic constituents are not. This
would explain ihv disturbance in the enzymatic coordi-
nation and the physiological weakness observed in the
inverted specimens.
BIBLIOGRAPHY
BOYCOTT, A., DIVER. C, HARDY. S. and TURNER. F.. Proc.Roy.8oc:
B.. !()'/. 152, 1929.
BRUCKNER, V. and IVANOVICS, G., Z.physiol.Chem.. 2 ',7, 281, 1937.
CASTLE, E.. Proc.Xat.AcSci.U.S.A.. 22. 336. 1936.
CAUSE, G.F., Biol.BulL. Hi, 448, 1939.
GAUSE, G.F. and SMARAGDOVA, N.P., Am. Naturalist. 7 ;. 1940.
GERSBACH, A., Zbl.Bakt., Abt. I, .S,s, 97, 1922.
HASTINGS, E. and SAGEN, H., J.Bact.. 2.), 39, 1933.
Si/'lh'AL TWIST A\D O/'T. At'TH ITY 97
JOHN-BROOKS, R., System of Bakteriol., 1, 104, 1930.
KIESEL. A.. EFIRIOCHKINA, E. and RALL, J., C.r.Acad.Sci.U.S.S.R.,
25, 481, 1939. '
KOLTZOFF, N., Biol.J. (Russian) ,!, 420, 1934.
LEWIS, J., J. Bad., 2',. 381, 1932; 25, 359, 1933.
LUDWIG, W.. Das Rechts-Links Problem. Berlin. 1932.
, Yer1i.Zool.Ges.. -iS, 21, 1936.
NEEDHAM, J., Nature. W,, 277. 1934.
OESTERLE, F., ZhL Bakt.. Abt. II, 7.';, 1, 1929.
PRINGSHEIM, E. and LANGER, J.. ZM.Bakt., Abt. II, 61, 225, 1924.
ROBERTS. J., Science. 87, 260, 1938.
STAPP. L. and ZYCHA. H., Arcli.Mikrohiol.. 2, 493, 1931.
ZVETKOFF, B., BuU.Soc.Natur. Moscow, 1938.
chapt?:r V
ANALYSIS OF VARIOUS BIOLOGICAL PROCESSES
BY THE STUDY OF THE DIFFERENTIAL
ACTION OF OPTICAL ISOMERS
Asymmetric Analysis. When one analyses the action
upon protoplasmic functions of dextrorotatory and of
laevorotatory isomers of various organic substances, one
often notices a difference in the effectiveness of the two
isomers. The existence or the absence of such a dif-
ference, as also its quantitative value, are evidently re-
lated to the physical structure and the chemical compo-
sition of protoplasm. One can, therefore, study the
mechanism of various biological processes by examining
how they are influenced by optical isomers of various
substances. It is thought that this method of analysis,
which we call ''Asymmetric Analysis," could contribute
to the clarification of several important problems of
comparative physiology.
With the idea of elaborating some systematic methods
of "asymmetric analysis", Gause and his associates have
undertaken the following investigations : 1. An analysis
was made of the mechanism of toxic action of optically
isomeric nicotines upon lower and higher animals. 2.
The mechanism of toxic action of optically isomeric or-
ganic acids upon lower and higher animals was similarly
studied. These two investigations will be reviewed and
discussed in the first section of this chapter. The study
of the effect of nicotine isomers in various animals led
to important observations on the evolution of the ner-
vous system. These observations will be discussed in the
second section. 3. A study was made of the action of
optically isomeric cinchonines upon various functions
of the cell. This study will be summarized in the third
section,
99
100 ASYMMETRIC ASALYSIS
SECTION I
ANALYSIS OF THE MECHANISM OF TOXIC
ACTION
A. TOXIC ACTION OF THE OPTICAL ISOMERS OF NICOTINE
A Case of Identical Mechanism of Action in the Two
Optical Isouters. Pictet and Eotscliv, in 1904, prepared
pure dextrorotatory (unnatural) nicotine, tested its tox-
icity on rabbits and guinea pigs, and ascertained that it
was less toxic than the natural 1-nicotine. They ex-
pressed the view, which subsequently was adopted by a
number of authors, of a different mechanism of toxic
action liy the dextro and by the laevo isomers, the symp-
toms of poisoning having been found different.
Maclit (1929) studied the pharmacological synergism
of stereoisomeric nicotines. He found that the toxic
action of a mixture of the 1- and dl- forms was stronger
than the additive action of these isomers. The con-
clusion that he reached then was that "an individual
cell may possess receptor groups of a laevo and dextro
type, and a mixture of two stereoisomers would thus have
a double point of attack in place of a single one, in case
only one of the optic isomers was used." The three fol-
lowing points in the work of Macht are open to criti-
cism. First, his important final conclusion is based on
a very small number of experiments. Then, the author
did not attempt to obtain a concentration-toxicity curve
which would allow one to make some quantitive calcula-
tions. Moreover, dl-nicotine is not the most suitable for
such experiments, as it usually contains some hydronico-
tine which influences the physiological effect (Gause,
1936) ; only the purest dextro nicotine obtained by re-
peated crystallizations with laevotartaric acid should be
used.
In the experiments of Gause and Smaragdova (1939)
the dextro isomer, in accord with the data of Pictet and
Eotschy, was found less poisonous than the laevo form.
A^O'MMirr/nc AXALYSf,^ 101
but tlu' relation ot* tlie increase in toxicity with the con-
centration was the same in the two isomers and a com-
plete identity of the temperature characteristics of toxic
action of the dextro and laevo forms was also observed
(in cold-blooded animals: fishes and tadpoles). The
identity of the relation of toxicity to concentration and
the identity of temperature characteristics are taken as
an indication of the identity of the mechanism of toxic
action of the two isomers. Both of them seem to act on
the same link in the system of physiological processes,
though with diiferent speed.
To illustrate these conclusions, let us consider in some
detail the results of recent experiments made with a brood
of the fish, Leuciscus idus var. orfiis (Gause and Smar-
agdova, 1939). The animals were placed in neutralized
solutions of nicotine of different concentrations, pre-
pared with redistilled water, and the killing time in sec-
onds was recorded. Figure 11 (upper part) represents
the relation of killing time to the concentration of nico-
tine.
It is to be pointed out that in the calculation of the
relative toxicity of 1- and d- nicotine one cannot use indis-
criminately results taken at various arbitrarily chosen
concentrations. The relative efficacy of the isomers
changes with change in absolute concentration. For com-
parison of the physiological effect of the optical isomers
one has to employ such characteristics of corresponding
curves of toxicity which are determined not by any
values of the absolute concentration of the poison but
by some physiological action. The most convenient is to
take the minimal lethal concentration of the poison (the
constant n, cf. formula below). In 1-nicotine fh = 0.0022% ;
in d-nicotine >/,i = 0.0064%. The coefficient of relative toxic
action (a) = y?d/>^i, which indicates how nuicli the 1-isomer
is more powerful than the d-isomer, is 2.91. (This co-
efficient will be called, hereafter, the "stereo-coeffi-
cient.")
102
ASYMMETRIC ANALYSIS
C 500
(U
E
'*- 3 0
_c
^ 2.5
6)
o
2 0
0 010%
0 02 0%
CONC.
10 lb 20 25
Log. of effective concentration i.v-ii)
Fig. 11. Killing action of the optical isomers of nicotine on the fish
Lencisciis idiis. The lower graph represents the toxicity curves in
logarithmic coordinates. (From Cause and Smaragdova, 1939.)
According to the principles of quantitative toxicology,
concentration-toxicity curves can usually be expressed
by the empirical equation of Ostwald :
k
^' ^ (x-n) ™
where u is the killing time, x the concentration of the
poison, n its minimal lethal concentration, and k and m
are constants. The constant m shows how rapidly the
toxicity increases with the concentration; it thus charac-
terizes the dynamics of the killing process. If one plots
log y on ordinates and log {x-n) on abscissae, the relation
between these variables will be represented by a straight
line. The slope of this straight line is measured by the
constant m.
ASYMMETRIC ANALYSIS 103
The lower part of Figure 11 represents the data on the
toxicity of dextrorotatory and laevorotatory nicotines
for Leuciscus, plotted in the manner just indicated. It is
evident that the slopes of the straight lines, characteriz-
ing the dynamics of the increase of toxicity with concen-
tration, are identical. It is therefore reasonable to con-
clude that in these experiments the mechanism of killing
action in the two optical isomers of nicotine is identical
in the sense defined. The unnatural dextro nicotine is
weaker only in the sense that a higher dose is required to
attain killing. Similar results were obtained also in ex-
periments with birds {Acanfhis flammea), lizards {La-
certa viridis) and tadpoles {Bana temporaria). Xo dif-
ference in the symptoms of poisoning by the two optical
isomers was observed.
Investigations were then carried out to determine the
temperature coefficients of toxicity of dextrorotatory and
laevorotatory nicotine for various animals. It is known
that if one plots the logarithms of the killing rate against
reciprocals of absolute temperature, one usually obtains
a linear relation. The slope of this straight line is gen-
erally represented by |li, which is known as the tempera-
ture characteristic (cf. Crozier, 1924). This characteris-
tic shows how the killing process is speeded up by the
rise of temperature. Physiological processes of differ-
ent nature, i.e., in which ditferent mechanisms are at play,
usually possess different temperature characteristics.
The temperature characteristics of toxicity for tadpoles
and for the fish Leuciscus were determined according to
the following procedure. Two solutions of dextro and
laevo nicotines were placed in a constant temperature
bath. After the temperature equilibrium was attained, a
number of fish or of tadpoles were immersed in the ves-
sels and the killing time was recorded. Figure 12 shows
the killing rate ( a value inverse to the killing time in
seconds) in the fish Leuciscus, due to the action of optic
isomers of nicotine at ditferent temperatures, Approxi-
104
AH \M METRIC ANALYSIS
^ 0.020 -
o
c
0.010
0)
o
c
o
o
2.0 -
0.00340
0.00350
Reciprocal of absolute temperature
Fig. 12. Effect of temperature on the killing rate of the fish Lciicis-
ciis idus by the optical isomers of nicotine. The lower graph represents
the killing rate plotted logarithmically. (From Cause and Smarag-
dova, 1939.)
mately isotoxic concentrations of the isomers, i.e., 0.007%
for 1-nicotine and 0.014% for d-nicotine, were used. The
temperature characteristics of the two isomers are prac-
tically identical: in the d-form \i = 37,500, and in the
1-form pi = 36,800. Such an identity of temperature char-
acteristics was also observed in experiments with tad-
poles; in the d-nicotine \x = 14,400, and in the 1-nicotine
|ii ^ 14,600. Hence the relation of toxic action to temper-
ature strongly supports the view that the mechanism of
toxic action is identical in the two optically isomeric
nicotines.
The same relations, that is, (1) a high toxicity of the
natural isomers, (2) the same relation between the in-
.1 .s r .1/ 1/ r/rinc asal vms
105
crease in toxicity and the concentration, and ( .'5) tlie same
temperature characteristics for the two isomers, have
been ol)served by Gaiise and Smarag'dova (ll)o8, ]9o9)
with nicotine on vertebrates (natural == laevorotatory),
with tartaric acid on fishes (natural = dextrorotatory),
and with cinchonine on ])aramecia (natural = laevoro-
tatory. Figure 13 shows that the dynamics of toxic action
are identical for dextrorotatory and for laevorotatory tar-
taric acids on the brood of the fish Lehistes reticulatus.
The temperature characteristics of toxic action for dex-
trorotatory tartaric acid was found to be 10,200 (cf.
02 04 06 08 10 12
Log. of effective concentration i.v-ii)
Fig. 13. Killing action of the optical isomers of tartaric acid on the
fish Lehifites reticulatus. The lower graph represents the toxicity
curves in logarithmic coordinates. (From Gause and Smaragdova,
1938.)
106
ASYMMETRIC ANALYSIS
Fig. 14) and for laevorotatory tartaric acid 9,700, in otlier
words, they were of the same order of magnitude. Sim-
ilar data were obtained also in experiments with the
brood of another species of fish, Platypoecilus maculatus
( Gause and Smaragdova, 1938).
^ 0.00200
10
o
I-
en
c
000100
o
4-
o
i_
en
_c
M-
O
o
3.3
3.2
3.1
3.0
2.9
2.8
L
Ji = 9.700
-L
0.00330
0.00340
Reciprocal of absolute temperature
Fig. 14. Effect of temperature on the killing rate of the fish Lebistes
retiCKlatiis by the optical isomers of tartaric acid. The lower graph
represents the killing rate plotted logarithmically. (From Gause and
Smaragdova, 1938.)
ASYMMETRIC ANALYSIS
107
Further, in experiments on the toxic action of opti-
cally isomeric einchonines upon paramecia, it was found
that the laevorotatory isomer inhibits the mechanism of
ciliary movement more rapidly than does the dextro form
(Gause, Smaragxlova and Alpatov, 1938). The dynamics
of toxic action in dextro and laevo einchonines were found
to be identical (cf. Fig. 15). A study of the temperature
characteristics of toxicity has also shown that these are
practically identical, 14,200 in the laevorotatory and
14,000 in the dextrorotatory isomer (cf. Fig. 16).
14 16 I 8 2 0 2 2
Log. of effective concentration {.v-)i)
Pig. 15. Killing action of the optical isomers of cinchonine on
Paramecium caiulatum. The lower graph represents the toxicity curves
in logarithmic coordinates. (From Gause, Smaragdova and Alpatov,
1938).
108
ASYMMETRIC ANALYSIS
B. TOXIC ACTION OF THE OPTICAL ISOMERS OF ORGANIC ACIDS
1. A Case of Different Mechanism of Action of the Two
Optical Isomers. In the cases reported so far the natural
isomer was more powerful in its physiological action than
the unnatural, the relation of increasing toxicity to con-
0.00330 000340
Reciprocal of absolute temperature
Fig. 16. Effect of temperture on the killing rate of Paramecium
caudatum by the optical isomers of cinchonine. The lower graph rep-
resents the killing rate plotted logarithmically. (From Gause, Smar-
agdova and Alpatov, 1938.)
ASYMMF/rRIC . 1 A 1 /> ) N/N'
109
centration and the temperature characteristics of toxic
action were identical for tlie two isomers. There are cases
in which it seems that none of these relations hold. GaUvSe
and Smarag-dova (1938) reported this situation in the
action of malic acid on the brood of two species of vivi-
parous fish, Lehistes rrticidafus and Platypoecilus macii-
latiis.
In these experiments they compared the natural laevo-
rotatory with the racemic malic acid (the significance of
the use of a racemate will be indicated below). It was
found that the natural laevorotatory malic acid is less
toxic than the racemic. The toxic action of weak
(0.05%) solutions of the laevorotatory and racemic malic
acids on Lehistes reUculatus at different temperatures
(16% 18% 21°, 26°, and 31°) is recorded in Figure 17. It
is quite apparent that the temperature characteristics of
c
o
u
0)
(1)
E
0)
o
en
c
o
en
o
1300
•
\
HOC
•
\
\
\
900
■
X
\
700
■
X
500
■
^
N^
3.4
I6»
21* 26*
3I»C
3.2
.
*^
3.0
■
>«=I2;300^
\M-9,3A.Q
2.8
^C^
0.00330 0.00340 O00350
Reciprocal of absolute temperature
Fig. 17. Effect of temperature on the killing time of the fish
Lehistes reticulatus by the optical isomei-s of malic acid. The lower
graph represents the killing rate plotted logarithmically. (From Gause
and Smaragdova, 1939.)
110 ASYMMETRIC ANALYSIS
toxic action are different in the racemate and in the laevo-
rotatory isomer. At temperatures from 16° to 26° C the
racemate is more toxic than the laevorotatory isomer,
whereas at the temperature of 31° C the latter is rela-
tively more toxic than the racemate. The temperature
characteristics of toxic action are also quite different. In
the laevorotatory isomer n = 12,300, and in the racemic
form |Li = 9,340.
In similar experiments with the fry of Platypoecilus
maculatus results of the same kind as those obtained with
Lebistes were recorded (cf. Fig. 18). The temperature
characteristic of toxic action of the left isomer of malic
acid is 16,930 and that of the racemic form 12,880.
The results obtained with fish were duplicated in a
study of the action of optically isomeric malic acids on
tadpoles of Rana temporaiia (Gause and Smaragdova,
1939).
Some experiments on the action of l(-) and of d( + )
leucine on the yeast Torula utilis (Gause and Smarag-
dova, 1938) also bring confirmatory evidence that the
unnatural form d(-|-) exerts a stronger action than the
natural and that their effect is of different nature.
Concerning the experiments in which racemic malic
acid was used, it should be mentioned that, in dilute
aqueous solutions, the racemic acid is completely disso-
ciated into dextrorotatory and laevorotatory constituents
(Ostwald, 1889). Therefore the greater biological activ-
ity of the racemate must probably be attributed to a
higher toxicity of the unnatural dextrorotatory compo-
nent.^
If our last assumptions are correct there would be a
series of cases in which, contrary to what has been de-
scribed above, the unnatural optic isomers are physio-
logically more effective and in which the mechanism of
lit is to be remembered that the natural dextrorotatory tartaric acid
and the natural laevorotatory malic acid belong to the same steric
series.
ASYMMETRIC ANALYSIS
111
0.00 300 0.00350 0.00400
Reciprocal of obsolute temperature
Fig. 18. Effect of temperature on the killing time of the fish
Platypoecilus maculatus by the optical isomers of malic acid. The
lower graph represents the killing rate plotted logarithmically.
112 ASYMMETRIC ANALYfilS
action of the two optic isomers is ditTerent. One could
not, then, speak of a single receptive protoplasmic sub-
stance which would simply react to a ditferent degree to
the two isomers, as is probably the case when the mech-
anism of action of the two isomers is the same.
2. Dual Activity of Organic Acids. The mechanism of
toxic action of the optical isomers of organic acids can be
also investigated from another point of view. Heilbrunn
(1928), among others, called attention to the dual nature
of the action of organic acids upon living systems: (1)
Organic acids produce an electro-chemical effect upon
the surface of the cells, primarily due either to a destruc-
tion of the negative charge of the cell surface by posi-
tively charged hydrogen ions or to other physico-chem-
ical surface 'pJienomena; (2) Owing to their relatively
weak electrolytic dissociation, the solutions of organic
acids contain a considerable proportion of non-dissociated
molecules which penetrate into the interior of the cells
where they produce transformations of a chemical nature.
Koltzotf's experiments (1915) on the action of ditferent
acids on the feeding activity of fresh-water vorticellids
furnish an example of the first type of action. There the
biological effect of the organic acid depends only on the
pH and the mechanism of this action consists in electro-
chemical changes upon surfaces directly accessible to hy-
drogen ions. The sinniltaneous occurrence of the first
and second type of effects is illustrated in the experi-
ments of Stiles and Rees (1935) who showed that the
killing action of monobasic organic acids of the aliphatic
series first diminishes with the elongation of the chain
of carbon atoms in the molecule, then reaches a minimum
with valeric acid and finally again increases with the fur-
ther elongation of the chain. This phenomenon was ex-
plained on the idea that the degree of electrolytic disso-
ciation diminishes with the increase in the weight of the
molecule, while the killing action of the non-dissociated
molecules increases with the increase of molecular weight.
ASYMMETh'W ANALYSIS 113
The observed lethal action, which is the resultant of the
partial lethal action of hydroo^en ions and of that of non-
dissociated molecules, would then decrease first and in-
crease afterwards as we indicated. Other investigations
on the mode of action of organic acids have been sum-
marized by Lepeschkin (1937).
Since optical isomers have all their physical and chem-
ical properties identical, except those which are directly
related to their structural configuration, one will observe
that, if the common properties only are involved in the
killing mechanism, the two isomers should produce the
same effect, while, if the properties which are ditferent
in the two isomers are involved in the killing action, the
two isomers will produce a different effect. It is further-
more assumed that the properties which are specific to
each isomer will be involved in the interaction of these
isomers with the protoplasm itself, within the cell, in op-
tically active medium, while the properties common to
the two isomers, such as the electric charge, the electric
conductivity (observed by Ostwald, 1889, to be the same
in the isomers of tartaric acid), the osmotic pressure,
etc., will be involved in such processes as conduction to-
ward the protoplasmic matter itself. Consequently, if
our assumptions are correct, when solutions of dextroro-
tatory and of laevorotatory acids are equally toxic for a
given animal, one may infer that the killing results from
physico-chemical injuries concerned with conduction or
the like. If, on the other hand, the two optical isomers
are not equally toxic, it is natural to think that the sur-
face effects just described could not induce death, so that
non-dissociated molecules have time to penetrate inside
the cells and there carry out their stereo-specific destruc-
tive actions.
With these ideas in mind, Gause and Smaragdova
(1938) determined the coefficient of relative toxicity of
the optical isomers of tartaric acid on various fresh water
animals. Some of their results are given in Table 18.
114 ASYMMETRIC ANALYSIS
TABLE 18
Coefficients of Relative Toxicity of Dextbokotatory Tartaric Acid
AND Significance of Differences in Relative Toxicity in
Different Groups of Animals.
(From Gause and Smaragdova, 1938.)
(M is the coefficient of toxicity; P.E. is the probable error.)
(1) Protozoa
M±P.E. 0.981±0.019
(2) Worms (3) Crustacea (4) Pisces
1.048±0.010
1.064±0.010 1.305±0.011
The figures represent the mean vahie (M) of the data for
all the animals of a given phylmn. The probable error
(P.E.) from the mean is also given.
The coefficient of relative toxicity in Protozoa is close
to unity, which means that the dextrorotatory and laevo-
rotatory tartaric acids are equally effective. On the con-
trary, in fishes, the optical isomers of tartaric acid dif-
fer strongly in their killing power, the stereo coef-
ficient being 1.305. The other groups of invertebrates
investigated occupy an intermediate position between the
protozoa and the fishes in their differential sensitivity to
the two isomers, the coefficient of relative toxicity reach-
ing 1.0-1-8 in the worms and 1.064 in the Crustacea.
Similar results were obtained also with the optical
isomers of malic acid.
According to the assumptions made, these data would
show that, in the killing of lower animals by tartaric
and malic acids, there predominates some electro-chem-
ical surface injury, while in higher animals internal chem-
ical injuries caused by non-dissociated molecules would
occur.
Cushny (1903, 1926) called into question the obser-
vations on the differences in the killing power of the dex-
trorotatory and the laevorotatory isomers of tartaric acid
in vertebrates, because the weak specific action of tar-
taric acids might, according to his opinion, be totally
concealed by the more powerful effect of the hydrogen ion
ASYMMETRIC ANALYSIS 115
concentration which is known to be identical in tlie solu-
tions of both optic isomers. But the objections of Cushny
must be considered in the light of the following recent
observations : 1. It is at present doubtful that the ef-
fect of hydrogen ions is always dominant over the spe-
cific action of non-dissociated molecules of organic acids
(cf. Gause, 1936), 2. Furthermore, Sizer (1937), in a
work on the stimulative effect of organic acids on various
animals has shown that Balanus halanoides is more sus-
ceptible to the action of hydrogen ions, while in Fimdulus
heteroclitus the effect of these ions does not predom-
inate over the specific action of nondissociated mole-
cules.
There is another essential point in the investigations
of Gause and Smaragdova. If the animals studied are
arranged in the order of increasing difference in the
toxic jDOwer of the two optical isomers : Protozoa
< Worms < Crustacea < Pisces, one obtains the phylo-
genetic series of gradually increasing differentiation.
This is not surprising if one considers the fact of the
progressively diminishing relative vital importance of
the physico-chemical injury of integuments when one
ascends the animal series. The nature of susceptible
integuments, the injury of which, according to our as-
sumptions, brings about death in lower animals, is not
known. It is possible that the respiratory surfaces are
among the most susceptible. In Protozoa the whole sur-
face of the cell is the respiratory surface. When one
ascends the animal series, respiratory surfaces become
more localized and more differentiated and the physico-
chemical injury of these surfaces progressively dimin-
ishes in magnitude as a cause of death. The results of fur-
ther investigations along this line have recently been pub-
lished by Gause and Smaragdova (1939).
116 ASYMMETRIC ANALYi^hS
SECTION II
ANALYSIS OF THE EVOLUTION OF THE
NERVOUS SYSTEM
1. Stereo-coefficients of Action of the Optical Isomers
of Nicotine in the Phijlogenetic Series. Since the two
optical isomers of nicotine exert their killing action by
the same mechanism but with a different strength one
can, by measuring this difference of potency in various
animals, study the properties of the specific receptive
substance in different species. In higher animals, as has
been already recorded above, there is some specific sen-
sitive substance which is affected to dift'erent degrees by
the toxic action of the dextro and the laevo isomers of
nicotine. Protozoa do not possess, as some observations
have shown, such a sensitive substance, and the dextro
and laevo isomers of nicotine are for them equally toxic.
The question arises of the nature of this specific sub-
stance and of the stage of evolution at which it first
appears.
Greenwood, as early as 1890, carried out an extensive
comparative investigation on the action of connnon laevo-
rotatory nicotine on invertebrates, attempting to establish
a parallelism between the toxic eifect of this alkaloid
which affects, as is known, the nervous system of ani-
mals, and the evolution of the nervous system. On the
basis of purely qualitative observations he reached the
conclusion that ''the toxic effect of nicotine on any or-
ganism is determined mainly by the degree of develop-
ment of the nervous system. Thus for Amoeba the sub-
stance cannot be regarded as exciting or paralysing ; it is
rather inimical to continued healthy life. As soon as any
structural complexity is reached, the action of nicotine
is discriminating in such a fashion that the nervous ac-
tions which are the expression of automatism, that is,
which imply coordination of impulses, are stopped first.
This is seen dimly in Hydra, and it is more pronunced
among the medusae. When structural development goes
ASYMMKTIi'lC AX ALT SIS 117
farther, the selective action of nicotine is traced readily,
as for example in PaJaeniou". Greenwood writes fui'ther
that: ''Animals which have enough in common to stand
near each other in classification, may yet react differently
to nicotine, each according to what I may perhaps call
its own balance of organisation."
Gause and Smaragdova (1939) made quantitative de-
terminations of the toxic action of the two optically
isomeric nicotines. The advantage of the use of the two
isomers will appear in the discussion of the results.
Experiments on vertebrates showed that the stereo-
coefficients of toxic action of optical isomers of nicotine
(a) are of the same order of magnitude in all ;ininials
studied :
Bird (AcanfJtis flaminra) a = 3.1
Lizard {Lacerta viridis) a = 2.4
Tadpoles {Rana temporaria) a = 3.0
Fish (Leuclscus idus) a = 2.9
Fish {Lebisfes reticulatus) a = 2.4
Mean a = 2.8
Inasmuch as the mechanism of toxic action is identical
in optically isomeric nicotines, one can, by the difference
of their effects, judge of the difference in spatial proper-
ties of the specific receptive substance assumed. As the
difference of effects remains constant, one can conclude
that the chemical nature of the receptive substance in
the vertebrates also remain essentially constant.
Since the procedure for the introduction of nicotine
was not the same for all the animals used — the poison
was introduced in the muscle of Lacerta while Lehistes
were immersed in the solutions of nicotine — the identity
of the stereo-coeffcient is an experimental proof that the
conditions of the penetration of nicotine do not affect
significantly either the mechanism of toxic action or the
reaction of the specific receptive substance.
Furthermore, the absolute sensitiveness to nicotine
in Acanihis is considerably higher than in Lacerta (0.8
118
ASYMMETRIC ANALYSIS
mg. per 100 gr. of weight as compared to 5.6 mg. per 100
gr.), but, practically, this difference in sensitiveness does
not influence the stereo-coefficient. The constancy of the
latter, despite a different sensitiveness, is also significant
in the study of the properties of the specific receptive
substance.
The results of investigations on the toxic action of the
two optical isomers of nicotine on fresh water and marine
invertebrates are given in Table 19. One sees that all
the invertebrates examined by Gause and Smaragdova
can be divided into two groups. The first includes the
animals for which the dextro and laevo isomers are equal-
ly toxic, and for w^hich the toxicity curves of the two
isomers fullv coincide. The second includes the organ-
TABLE 19
Comparative Killing Action of the Optical Isomers of Nicotine on
Invertebrates
(Tiie sign = means that d and 1 nicotines are equally toxic; the coeffi-
cient (X indicates to what degree 1 is more toxic than d nicotine.)
Animal
Comp.
Toxicity
Animal
Comp.
Toxicity
Protozoa
Annelida
1. Paramecium cauda-
15.
Saccocirrus papillo-
tum
cercus
a — 2.3
2. Euplotes patella
16.
Perinereis cultrifera
a -1.9
3. Stentor coeruleus
17.
Arenicola grubii
a >1
4. Spirostomtim ambi-
18.
Pristina longiseta
a— 2.09
guum
19.
Limnodrilus hoff-
Coelenterata
meisteri
a— 3.45
5. Hydra fusca
—
20.
Helobdella stagnalis
a— 4.0
6. Cladonema radiatum
21.
Nais C07nm.unis
a =2.41
Platyhelminthes
22.
Chaetogaster langi
a — 3.13
Turbellaria
23.
Stylaria lacusti'is
a >1
7. PolyceUs nigra
, ,
24.
Aelosoma variegatum
a -1.76
8. Phaenocora sp.
25.
Aelosoma hemprichi
a -1.84
9. Dalyellia brevimana
Chaetognatha
10. Procerodes lohata
26.
Sagitta setosa
a— 2.7
11. Leptoplana tremel- ■
Arthropoda
laris
27.
Daphnia magna
Rotatoria
28.
Cyclops serrulatus
12. Euchlanis triquetra
29.
Gammarus marinus
13. Rotifer vulgaris
30.
Drosophila melan-
Nemertinea
ogaster
14. Lineus lacteus
(2-days old larvae were
immersed in nicotine
solutions.)
ASYMMETRIC ANALYSIS 119
isms in wliicli the laevo isomer of nicotine is more toxic
than the dextro- isomer. Tlie two groups correspond to
large divisions of the animal kingdom, and witliin each
division, there are hardly any exceptions.
All the representatives of Protozoa, Coelenterata, Tur-
bellaria, Rotatoria and Nemertinea studied belong to the
first group. They are devoid of spatially specific recep-
tive substances in the process of poisoning by nicotine.
It should be mentioned that the stereo-coefficients in in-
vertebrates are not atfected by the differences in the ab-
solute sensitiveness to nicotine, exhibited by various spe-
cies, as it has been noticed in vertebrates. Thus, for ex-
ample, Leptoplana is considerably more sensitive to nico-
tine than Procerodvs, but both these turbellarians are
characterized by an equal effect of the dextro and laevo
isomers. There are many other examples of the inde-
pendence of these characters.
The lowest groups in the phylogenetic series, in which
a stronger eifect of the laevo isomer of nicotine is ob-
served, are the annelids, and particularly the Archian-
nelids {Saccocirrus), the Polychaeta and the Oligochaeta
and the primitive representatives of Deuterostomia {Sa-
gitta setosa). In Arthropoda (Crustacea and Insecta)
this etfect is absent, an equal toxicity of the dextro and
the laevo isomers is again observed.
Let us now compare the stereo-coefficients in verte-
brates and in those invertebrates which show a higher
sensitivity to the laevorotatory nicotine. The following
values were recorded in invertebrates :
Saccocirrus papillocerciis
Perinereis cult rif era
Pristina longiseta
Limnodrilus Jiofmeisteri
Helobdella stagnalis
Nais communis
Ch act og aster langi
Aelosoma variegatum
Aelosoma hemprichi
Sagitta setosa
Mean a
a
2.3
a
1.9
a
—
2.1
a
=
3.4
a
4.0
a
—
2.4
a
3.1
a
1.8
a
1.8
a
2.7
a
2.6
120 AswiMtyriiw A\ALysii<i
The limits of error in measuring the coefficient a may
extend over a rather wide range. Thus the following
values were obtained in two independent measurements :
in Nais 2.57 and 2.25; in Liwiwdrilus 2.9 and 4.0; in
Aelosoma variegaium 1.79 and 1.74; in Aelosoma Jiem-
prichi 1.50 and 2.18. Nevertheless the order of magnitude
of the average is significant. We find nearly the same value
in invertebrates (2.6) and in vertebrates (2.8).
The data above mean that the Protozoa, Coelenterata,
Turbellaria, Rotatoria and Nemertinea are deprived of
the spatially specific receptive substance which responds
differentially to the left isomer of nicotine. Annelides,
Chaetognatha and Vertebrates possess this receptor,
while in Arthropoda it is absent again.
2. The Acetylcholine System and the Dlfferottial Ef-
fect of the Optical Isomers of Nicotine. Considering that
it is the nervous system in animals which is affected by
nicotine and that there is an identity of stereo-coeffi-
cient in invertebrates and in vertebrates, in spite of es-
sential differences in the morphology of their nervous
system, we come to the conclusion that there is some
uniform receptive substance distributed in the various
nervous systems of these animals. However, this chem-
ical constituent is not an obligatory component of every
nervous system; even some quite differentiated nervous
systems of lower invertebrates (Turbellaria and Nemer-
tinea) are deprived of it.
A study of the present views on the mechanism of nic-
otine toxic action will furnish more information on the
nature of the receptive substance. Thomas and Franke
(1924, 1928, 1933) have shown that it is the paralysis of
the peripheral neuro-muscular junctions of the respira-
tory muscles which is the cause of death of higher ani-
mals in acute nicotine poisoning. This view was con-
firmed by Gold and Brown (1935). We are thus led to
the old classical observations of Langley (1904) that in
the ''neuro-muscular junction" there is a certain sensi-
A.SYMMI-JTinv A^ALYSI^S 121
tive "receptive substance" wliicli is tlie tirst to be af-
fected by nicotme.
On tile other hand, since the cUissical works of Loewi,
it is known that, in the transmission of impulses from
nerves to effectors the various steps are as follows (1)
nerve impulse — ^ (2) chemical mediator — ^ (3) receptive
substance — v (-t) specific response (for literature see
Cannon and Eosenblueth, 1937). There are some indi-
cations that the chemical mediator in the voluntary mus-
cles of higher animals is acetylcholine. Its action on the
receptive substance in this case reminds one of that of
nicotine: in small doses it excites, and in larger doses it
paralyses, and according to the current views, nicotine,
in case of an acute poisoning, atfects in some irreversible
way the receptive substance, upon which acetylcholine
mediation is no more effective. In other words, nicotine
(at least in experiments of our type) acts upon neuro-
effector synapses of voluntary muscles. In its action it
reminds one of acetylcholine, the substance which trans-
mits the excitation in these synapses. Consequently the
receptive substance in nicotine poisoning has some close
relation to the receptive substance for chemical media-
tion.
The experiments just described permit one to divide
the animals into two groups according to the nature of
the receptive substance atfected by nicotine. It might
be that animals possessing a receptive substance differ-
entially atfected and those possessing a receptive sub-
stance identically affected by optically isomeric nicotines
ditfer also in their receptivity to the normal chemical me-
diator, and consequently in peculiarities of the transmis-
sion of nerve impulses.
An examination of the data on the distribution of
acetylcholine in different groups of invertebrates will
throw a new light on this problem. Despite the often
questionable findings concerning the presence of this
substance which is ascertained bv the action of extracts
122 ASYMMETRIC ANALYSIS
on different organs while not a single of the ordinarily
used organs is strictly specific, as Cannon and Rosen-
blueth (1937) pointed out, the results may be regarded
as sufficiently reliable if they are repeatedly observed
with several different procedures. The most extensive and
elaborate investigations were carried out by Bacq (1935)
at the Biological Station of Naples. He did not find
acetylcholine nor the enzyme which destroys it, choline-
esterase, in the tissues of different Coelenterates. The
muscles of Annelids and of lower Deuterostomia (Holo-
thuria) contained acetylcholine and choline-esterase. In
the muscles of Crustacea he found so little acetylcholine
that he concluded that the transmission of impulses
from the motor nerve to the muscle in these animals is
not accomplished by means of this mediator. He insisted
on this point at the conference devoted to this problem
held in Cambridge in 1937. On the other hand, there
are some preliminary communications by Nachmanson
(1937), according to which there is some choline-esterase
in the ganglions of Crustacea. What is certain, however,
is that neuro-effector synapses of the muscles of Crus-
tacea are not typical acetylcholine systems, if only for
the reason that they are extremely insensitive to the
action of externally applied acetylcholine.
In the accompanying table w^e compare the observa-
tions of Bacq with those of Gause and Smaragdova. In
six animal groups the two series of independently obtain-
ed results coincide. If our suggestions are correct, the
differential killing action of optical isomers of nicotine
could be employed to detect the presence of the specific
receptor characteristic for the acetylcholine system in
the neuro-effector synapse of voluntary muscles (Gause
and Smaragdova 1939). But further investigations are
necessarv for a final conclusion on this problem.
A S 1 -1/ .1/ /•; 77.' / (' - 1 XALYSIS
123
TABLE 20
Comparative SmiY, of thic Presence in Various Animals of a Stereo-
Differential Toxic Action of the Optical Isomers of Nicotine
AND of A( KTYLCIIOLINE MEDIATION IN THE TRANSMISSION
OF Nerve Impulse
Animals
Stereo-Differential
Action of Nicotine
(Gause and Smarag-
Acetj'lcholine
Mediation
(Bacq. 1935)
dova,
1939)
1.
Coelenterata
Absent
Absent
2.
Annelida
Present
Present
3.
Lower Deuterostomia
(Holothuria
for ace-
tylcholine and
Chaetog-
natha for nicotine)
Present
Present
4.
Crustacea
Absent
Absent
5.
Insecta
Absent
Absent
6.
Vertebrata
Present
Present
SECTION III
ANALYSIS OF THE MECHANISM OF A^AEIOUS
PHYSIOLOGICAL FUNCTIONS IN PROTOZOA
The following attempt at aii ''Asymmetric Analysis"
of physiological functions in protozoa is based on the
fundamental principle of dissociability of physiological
processes. The action of the optical isomers of some
organic substance will be studied on some particular
function and the stereo-coefficient of action determined
for that function. From the similarity or dissimilarity
of the coefficient various conclusions can be drawn on the
nature or mechanism of the function.
It should be noticed that a similar method is followed
in the temperature analysis of biological processes (cf.
the recent discussion of this subject by Hoagland, 1935).
"When two separate processes reveal different tempera-
ture relations it is believed that they are not directly
controlled by some common ''master reaction". (Con-
cerning the caution with which the notion of "master
reaction" should be used, cf. Burton, 1936 and Hoag-
land, 1937.)
124 Al^Y.}[}[ErRIC ANALYSLS
The experiments of Gause, Sniaragdova and Alpatov
(1938) to be reported here, consisted in the analysis of
the action of the optical isomers of cinchonine on the
rate of the following functions of the infusorian Para-
mecium caudatiim: (1) The feeding rate, as measured by
the number of food vacuoles formed in water suspensions
of india ink; (2) The velocity of expulsion of gastric
vacuoles; (3) The division rate; (4) The velocity of loco-
motion in thin glass tubes, according to the method of
Glaser (1924) ; (5) The death rate, death being diagnosed
by the complete cessation of all motion.
It was found that the stereo-coefficient of action of
cinchonine (a) was of the same order of magnitude (about
1.3) in the case of the inhibition of the following three
functions : the formation of the gastric vacuoles, the ex-
pulsion of these vacuoles, and the division rate. In other
words cinchonine inhibits some susceptible system which
controls these three functions. The gastric vacuoles in
paramecia separate from the gullet, being, so to say,
pulled away by the active protoplasm (cf. Metalnikoff,
1910 and Bozler, 1924). Their formation and their ex-
pulsion are evidently connected with the degree of activ-
ity of the protoplasm. These two functions, as Avell as
the rate of division, are, therefore, expected to be con-
trolled by the rate of metabolism in the endoplasm of the
Paramecium. In all probability optically isomeric cin-
chonines, inhibiting one of the phases of metabolism in
the endoplasm, depress all the three functions together.
On the other hand, the cessation of the motion in para-
mecia by cinchonine is evidently connected with the pois-
oning of the svstem of locomotory cilia. This svstem is
localized in the ectoplasm (cf. Kalmus, 1931). The stereo-
coefficient of action of the optical isomers of cinchonine
is significantly ditferent from that previously recorded, it
reaches 1.98 (the left isomer being, as before, more pow-
erful than the right). These data are presented in
Table 21.
yi^'vi/i//;77.'/r .i,v.i/.r.s7.s' 125
TABLE 21
Stereo-Coefficients of Action of the Optical Tso.mkus of Cinchoxine
ox Vauioi's Functions in Paramecium caudatum.
(Froji Gaise, Smaragdova and Alpatov, 1938.)
Function Stereo-coefficient
Inhibition of f 1. Formation of gastric vacuoles j ^ =: 1.36
endoplasm ■{ 2. Release of gastric vacuoles
[ 3. Division rate
ot = 1.36
a = 1.24
Inhibition of \
ectoplasm ( 1. Mechanism of ciliary motion fi = ^-^^
The differences ol)served in the vakie of the stereo-
coefficient suggest that, in the ectophism, cinchonine in-
hibits a receptive substance diiferent from that of the
endophism. A physiological differentiation of the cells
into ectoplasm and endoplasm would then be brought into
evidence in Paramecium caudatum by our method of
''asymmetric analysis".
However, in another species of paramecia {Para-
mecium hursaria), the stereo-coefficients of the action of
optically isomeric cinchonines on various functions did
not disclose such a dilfereuce; the same stereo-coeffi-
cient of inhibition was observed for the endoplasmic and
ectopia smic function.
For the study of the effects of the optical isomers of
cinchonine on the rate of movement of paramecia the
procedure Avas, in general, as follows. To 2 cc. of cin-
chonine solution of a given concentration 5 drops of a
culture of Paramecium caudatum were added. A little
quantity of the cinchonine solution with infusoria was
then transferred with a pipette into a thin glass tube
and the latter was placed on a graduated glass plate on
which the velocity of motion was measured with the aid
of a stop-watch. The determinations were made every
ten minutes for eighty minutes.
While, for a time, no significant ditference in velocity
could be observed in the control and in the right isomer
solution, the paramecia in the left isomer of the same
126 ASYMMETRIC ANALYSIS
strength i^reseuted a considerable increase in the rate of
movement. The left isomer of cinchonine definitely call-
ed forth at first a strong stimulation of movement; sub-
sequently the motion slowed down and finally the para-
mecia died. With the right cinchonine the stimulation
phase was entirely absent under all concentrations em-
ployed, only the inhibition phase could be observed.
So only the laevorotatory isomer has the specific power
of stimulating the ciliary movement. One can suppose
that the left isomer, because of peculiarities of its spatial
configuration, interacts with the system of reactions
which control the ciliary motion, while this system re-
mains as if "closed" for the dextrorotatory isomer. In
distinction from this stimulating effect, the less specific
process of toxic destruction of the locomotory force of
the cilia is carried out qualitatively in the same way by
both optic isomers of cinchonine, the rate of the reaction
only is different. This situation has its parallel in the
following observation of Krebs (1936). He has recently
pointed out that in the metabolism of amino acids some
specific transformations such as the splitting of the imi-
dazole ring in histidine (Edlbacher and Neber, 1934), or
the oxidation of the ring in tyrosine (Bernheim, 1935),
are open only to the natural amino acids of the left series
and are closed for the right forms. On the contrary, in
other less specific reactions, such as deamination, both
optic isomers of amino acids can participate.
Further data on the action of optical isomers of cin-
chonine upon various protozoa are given in the orig-
inal paper by Gause, Smaragdova and Alpatov (1938).
SUMMARY
1. The study of the mechanism of various biological
processes by examining how they are influenced by opti-
cal isomers of various substances is presented as a
method of investigation called '^ Asymmetric analysis."
This method is applied here in the study of (1) the
ASYMMETRIC ANALYSIS 127
meclianism of toxic action, (2) the evolution of the ner-
vous system, (3) the mechanism of various physiological
functions in protozoa,
2. The two optical isomers of a toxic substance may
exhibit different degrees of toxicity (the natural isomer
being- more toxic) but possess the same mechanism of
toxic action, as judged by the identity of the relation of
increasing toxicity to concentration and by the identity
of the temperature characteristics. Such conditions have
been observed, in particular, in nicotine. There are cases
in which none of the two relations just mentioned hold.
The last series of cases cannot be accounted for by the
assumption of a receptive substance diversely affected
by the two isomers.
3. The coefficient of relative toxicity of the two isomers
of tartaric acid increases from 1 to 1.305 when one
passes from the protozoa to the fishes through the worms
and the Crustacea. The killing action, in the lower forms,
seems, then, to be due to factors which are common to the
two isomers, while, in the higher forms, it is due to factors
which differ in the two isomers. It is suggested that the
factors of the first type are those which act mostly on the
surface of organisms, and the factors of the second type,
those which act internally. The problem of the mode of
action of toxic substances is then linked to that of the
evolution of the integuments in fresh water animals.
4. The study of the toxic action of nicotine in animals
of variously developed nervous systems points to the
absence of a spatially specific receptive substance in Pro-
tozoa, Coelenterata, Turbellaria, Rotatoria and Nemer-
tinea, and to the presence of such a substance in Annelids,
Chaetognatha and Vertebrates. In Arthropoda it is ab-
sent again. A comparison of its distribution with that of
acetylcholine in different groups of animals leads to sig-
nificant data on the evolution of the nervous system. The
receptive substance in nicotine poisoning shows some
close relation to the receptive substance for chemical
mediation in the transmission of the nerve impulse.
128 ASYM}fETRrC ANALYSTS!
5. The results of the toxic action of the optical isomers
of cinchonine on Paramecium caudatum bring into evi-
dence a difference in the physiological functions con-
trolled by the ectoplasm and those controlled by the en-
doplasm. Of the two isomers of cinchonine only the
laevorotatory showed the specific power of stimulating
ciliary movement.
BIBLIOGRAPHY
BACQ. Z., Erg. Physiol.. :i7, 82, 1935.
BERNHEIM, F., J. Biol. Chem., Ill, 217, 1935.
BOZLER, E., Arch. Protistenk., J,9, 163, 1924.
BURTON, A., J. Cell. Comp. Physiol.. 9, 1, 1936.
CANNON, W. and A. ROSENBLUETH, Autonomic Neuro-Effector Sys-
tems, New York, 1937.
CROZIER. W., /. Gen. Physiol., 7. 1924.
CUSHNY, A., J. Physiol.. SO, 176, 1903.
, Biological Relations of Optically Isomeric Substances, Bal-
timore, 1926.
EDLBACHER, S. and W. NEBER, Z. physiol. Chem.. 22Jf, 261, 1934.
GAUSE, G. F., Raumaufbau des Protoplasmas, Erg. Biol., l-l 54, 1936.
, Nature. ISS. 976, 1936.
, and N. P. SMARAGDOVA, Biol. Zhiirn.. 7, 1938; Phy-
siol. Zooh. hi. 1939; Zool. Zhurn., 18, 1939; Bull. biol. et med. exp.
Moscow, 7, 105, 108, 1939.
, and W. W. ALPATOV, Biol. Zhourn., 7, 763,
1938.
GLASER, O., J. Gen. Physiol., 7, 177, 1924.
GOLD, H. and F. BROWN, -/. Pharmacol.. J}, 463, 1935.
GREENWOOD, M., ,/. Physiol.. 11, 573, 1890.
HEILBRUNN, L.. The Colloid Chemistry of Protoplasm. Berlin, 1928.
HOAGLAND, H., J. Cell. Comp. Physiol., 10, 29, 1937.
KALMUS, H., Paramecium, Jena, 1931.
KOLTZOFF, N., Trans. Shaniavsky Univ. Biol. Lab.. 1, 189, 1915.
KREBS. H. A., Ann. Rev. Biochem.. .>, 247, 1936.
LANGLEY, N., J. Physiol., 33, 374. 1905.
LEPESCHKIN, W., Zell-Nekrobiose und Protoplasma-Tod. Berlin, 1937.
MACHT, D., Proc. Nat. Acad. Sci., U.S.A., 15, 63, 1929.
METALNIKOFF, S.,Trans. St. Petersburg Biol. Lab., 11, 3, 1910.
NACHMANSOHN, D., Nature. 1937.
OSTWALD, W., Z. physikal. Chem.. 3. 369, 1889.
PICTET, A and A. ROTSCHY, Ber. chem. Ges.. 37, 1225, 1904.
SIZER, S., Physiol. Zool., 10, 327, 1937.
STILES. W. and W. REES, Protoplasma. 22, 518, 1935.
THOMAS, J. and F. FRANKE, J. Pharmacol. 23, 150, 1924; 34. Ill,
1928; .'/8, 199, 1933.
APPENDIX
ASYMMETRY OF PROTOPLASM AND THE STRUCTURE
OF THE CANCER CELL
When, in 11I2:I, ()tto Warburg reijortod thai the oxyda-
tive metabolism of cancer cells is somewhat defective
and that growth and multiplication in cancerous tissue
is provided mainly by fermentative processes accompa-
nied by the formation of large quantities of lactic acid, it
was thought that the solution of the old mystery about
the nature of cancer had been found. Numerous at-
tempts of cancer therapy based on this principle, and
consisting mostly in inhibiting anaerobic processes in
malignant cells, were made. But all these attempts
failed. It was then learned that anaerobic metabolism is
not a characteristic feature of malignancy, it is observed
in many embryonic tissues.
In view of such failures, James Ewing, director of the
Memorial Hospital, New York, remarked before the Na-
tional Academy of Science (April, 1938), that the funda-
mental nature of malignant growth is probably an in-
solvable problem and that investigations on this prob-
lem have consumed much time and money without pro-
viding knowledge of practical value. There is, accord-
ing to him, urgent need for greater support of clinical
investigations which yield some practical results.
In spite of that skeptical attitude toward the pros-
pects of fundamental cancer research, the year 1939
brought forth an important discovery on the structure of
the cancer cell. Two distinguished Dutch chemists, Fritz
Kogl and Hanni Erxleben isolated from proteins of ma-
lignant cells the unusual optical isomer of glutamic
acid (of the right steric series), which never occurs in
proteins of healthy cells. This important finding was
rapidly followed by significant practical applications.
Waldschmidt-Leitz (1939) discovered in the serum of
cancer patients proteolytic enzymes with unusual stereo-
129
130 ASYMMETRY OF PROTOPLASM AND CANCER
chemical behaviour. These enzymes are absent from the
sermn of healthy persons.
Kogl and Erxlebeu also isolated several other amino
acids from proteins of normal and malignant tissues and
measured their optical rotatory power. Serine and pro-
line, which undergo easily a partial racemization in hy-
drolysis, were' obtained as partially racemic products in
healthy tissues. Proline instead of specific rotation
aa = ~ 84.9° gave a value of « = - 82.4°, and serine, in-
stead of a = H- 14.45°, gave a = + 8.38°. But such amino
acids as valine, leucine and glutamic acid were practically
optically pure when isolated from healthy tissues, while
partially racemic preparations were isolated from malig-
nant tissues (Table 1).
TABLE 1
Rotatory Powek of Some Amino Acids Lsolated from Ovarial
Carcinomes.
• (Kogl and Erxleben, 1939.)
Amino acid
Expected specific
rotation
Observed specific
rotation
Leucine
Lysine
Valine
Glutamic acid
+15.40
+14.60
+28.80
+31.70
+13.20
+13.50
+26.90
+ 4.60
In the case of glutamic acid, racemization is most evi-
dent, since as far as 42.7% consists of the unusual d(-)
form. Such observations have led Kogl and Erxleben to
conclude that the unusual optical isomers of some amino
acids participate in the composition of cancer cells. The
latter would, then, be characterized by some particular
spatial molecular configuration, on account of which the
growth-controlling enzymes would be disturbed.
According to Kogl and Erxleben, partial racemization
of glutamic acid in cancer tissues is most evident and it
could be checked easily. This observation has been se-
verely criticized by Chibnall (1939) and also by Graff
(1939) who reported to have isolated only optically pure
A8YMMETR Y OF PRO TO PLA^M AND CANCER 131
1( + ) giutainic acid from malignant cells. Kogl and Erx-
leben (1939) inmiodiately pointed out that their oppo-
nents did not pay sufficient attention to the different sol-
ubilities of the optical isomers. Racemic dl-glutamic
acid, in the form of both chlorhydrate and barium salt, is
two times more soluble than 1-glutamic acid. The pure nat-
ural isomer consequently cristallizes first and, if the crys-
tallization is not complete, the racemic isomer will be left
in the mother liquid.
Lipmann and his collaborators (1940) also opposed
their findings to Kogl's and Erxleben's data. On ac-
count of some difficulties in the ordinary isolation pro-
cedures, Lipmann attempted to determine the total d-
amino acid content of the human tumors and of normal
tissues by means of d-amino acid oxidase with the aid of
the Krebs enzyme. He found 1.85% of d-amino acids in
hydrolvzates of normal tissues and 1.84% of d-isomers in
those of cancer tissues, that is, practically the same value
in the two cases. However, Lipmann himself admits that
the accuracy of his method is not great. Moreover, since
each hydrolysis inevitably leads to a partial racemization
of such labile amino acids as serine and proline, the deter-
mination of total d-amino acid content loses some of its
significance.
On the other hand Kogl's data have been confirmed by
Arnow and Opsahl (1939). The glutamic acid which they
isolated from normal tissues had an optical rotation of
a — --h 31.0^, and that isolated from malignant tissue had
an optical rotation of a = H- 5.5°.
If one assumes that Kogl's data are correct, it is, how-
ever, not clear whether the unusual optical isomer of glu-
tamic acid pre-exists in the cancer cell, or whether the par-
tial racemization observed is of a factitious nature. If
glutamic acid, in the protein molecule of cancer cells, en-
ters into some special labile compound different from that
in which it exists in usual protein molecules, it is con-
ceivable that hydrolysis could lead to a partial racemiza-
tion in the cancer cell but not in the normal cell. This
132 ASYMMETRY OF PROTOPLASM A^D CANCER
possibility can be checked. Lipniaiiii reports that most of
his hj^drolyses were carried out in HCl containing* heavy
water. Subsequent determinations of the content of deu-
terium attached to the alpha carbon atom of glutamic
acid w^ill show, according to the suggestion of du Vig-
neaud whether or not the partial racemization is due to
the process of hydrolysis.
If the recent data of Waldschmidt-Leitz and Mayer
(1939) are confirmed, they will undoubtedly lead to new
developments in the study of the problem of cancer from
the viewpoint of protoplasmic asymmetry. According
to Waldschmidt-Leitz, if unusual optical isomers of some
amino acids really enter into the composition of malignant
cells, there should be specific proteolytic enzymes to cat-
alyze the spatially unusual metabolic processes. It is
known that peptidases of healthy animal tissues do not
split up such polypeptides which consist of unusual optic
isomers of amino acids. Waldschmidt-Leitz showed this
to be true also of the aminopoly peptidase and the dipep-
tidase from the serum of healthy persons. But the prop-
erties of peptidases from the serum of cancer patients are
radically diiferent, they can split up polypeptides con-
sisting of unusual optic isomers. This feature was used
by Waldschmidt-Leitz in his diagnosis method.
One can hope that more light be shed on this important
problem in the near future.
BIBLIOGRAPHY
ARNOW, E. and J. OPSAHL, Science, 90, 257, 1939.
BAYBRLE, H., Biochem. Z., 303, 251, 1939.
CHIBNALL, A., Nature, 14',, 71, 1939.
EWING, J., Science, 87, 399, 1938.
GRAFF, S., J. Biol. Cheni.. 130, 13, 1939.
KoGL, F. and H. ERXLEBEN, Z. physiol. Chem., 258, 57; 2G1, 154;
Naturiviss, 27, 486; Nature, 1. ',.',, Ill; Z. Krehsforsch., J,9, 291, 1939.
and A. AKKERMAN, Z. physiol. Chem., 262,
141, 1939.
LIPMANN, F. et at., Science, 01, 21, 1940.
WALDSCHMIDT-LEITZ. E. and K. MAYER, Z. physiol. Chem., 262, IV
1939.
WARBURG, O., Uber den Stoffwechsel der Tumoren. Berlin. 1926.
GENERAL BlI^LIOGRAPHY OF THE LITER-
ATURE ON THE ASYMMETRY OF
BIOLOGICAL MATERIAL
(In Chronolofjicnl Onh r)
This bibliograpliical list contains titles of publications
which concern the problem of the Asymmetry of Biologi-
cal Material, even if they were not mentioned in the text
of this book. On the other hand, papers cited in the book
but concerning- only indirectly our subject have been
omitted. The list includes the literature published up to
about the middle of the year 1940.
1824
Fresxel, A., Memoire sur la double refraction . . . Bull.
Soc.Pliilomat., p. 147.
1848
Pasteur, L., Recherches sur les relations qui peuvent
exister entre la forme cristalline, la composition chimique
et le sens de la polarisation rotatoire. Ann.Chim.Pliys.,
2',, 442-459.
1860
Pasteur, L., Note relative au Peniclllium glaucum et a
la dissymetrie moleculaire des produits organiques nat-
urels. C.R.Acad.ScL, 51, 298-299.
, Recherches sur la dissymetrie molecu-
laire des produits organiques naturels. Soc.Chim.Paris.
Legons de chiniie professees en 1860. English translation
in Alembic Club Reprints, No. 14, 1897.
1861
Pasteur, L., Lettre aux redacteurs. Anii.Chim. et Phys.,
61, 484.
1869
GixTL, W.F., tjber einige Bestandtheile von Fraxinus
excelsior. Journ.pract.Chem., JOG, 486.
133
134 GENERAL BIBLIOGRAPHY
1874
LeBel, J.A., Sur les relations qui existent entre les
formiiles atomiques des corps organiques et le pouvoir
rotatoire de leurs dissolutions. Bull.Soc.Chim,., 22, 337-
347.
van't Hoff, J.H., Sur les formules de structure dans
I'espace. Arch.Neerland., 9, 445. English translation:
The arrangement of atoms in space. New York, 1898,
1884
JuisTGFLEiscH, M.E., Sur le dedoublement des composes
optiquement inactifs par compensation. Bull.Soc.cJiim.
Paris, J,l, 222-233.
LippMAN, E., tJber das Vorkommen von Leucin und
Tyrosin in der Eiibenmelasse. Ber.chem.Ges., 11, 2835-
2840.
Pasteue, L., La dissymetrie moleculaire. Rev.Scient.,
J,, 2-6.
Wyrouboff, G., Discussion. Bnll.8oc.Chim. Paris, Jjl,
212.
1886
PiUTTi, A., Ein neues Asparagin. Ber.chem.Ges., 19,
1691-1695.
Schulze, E. and E. Bosshard, Untersuchungen iiber
die Aminosauren, welche bei Zersetzung der Eiweisstoffe
durch Salzsaure und durcli Barytwasser entstehen.
Z.physiol.Ghem., 10, 134-145.
1889
OsTWALD, W., Tiber die Affinitatgrossen organischer
Sauren und ihre Beziehungen zur Zusammensetzung und
Konstitution derselben. Z .physical. Chcm., 3, 369-422.
1890
FiscHEE, E., Synthese der Mannose und Liivulose.
Ber.chem.Ges., 2J,'370-394.
, Synthesen in der Zuckergruppe. Ber.
chem.Ges., 23, 2114-2141 ; also 27, 3189-3232, 1894.
GENERAL BIBLIOCRAPHY 135
1891
Nencki, M., Die isomeren Milchsaurcn als Erkennimgs-
mittel einzolnor Si)alt]nlzarten. ZhJ.Balt., 0, 304-306.
1893
Beowx, H. aistd H. Morris, A contribution to the chem-
istry and physiology of foliage leaves. J.Cliem.Soc.,
63, 604-677.
Chabrie, C, Sur la toxicite des acides tartriques ster-
eoisomeres. C.R.Acad.Sci., 116, 1410-1413.
Pere, a., Sur la formation des acides lactiques isome-
riques. Ann.Inst. Pasteur, 7, 737-750.
1894
Curie, P., Sur la symetrie dans les phenomenes phy-
siques. J.de Physique, 3, 393-415.
Fischer, E. and H. Thierfelder, Verhalten der ver-
schiedenen Zucker gegen reine Hefen. Berxliem.Ges., 27,
2031-2037.
Kayser, E., Etudes sur la fermentation lactique. Ann.
Inst. Pasteur, 8, 737-784.
1898
Bartrum, CO., Chance or vitalism! Nature, 58, 545.
Brion, a., tJber die Oxydation der stereoisomeren
Weinsauren im tierischen Organismus. Z .pliysiol.Cliem.,
25, 283-295.
Eerera, G., Asymmetry and vitalism. Nature, 58, 616.
Fischer, E., Bedeutung der Stereochemie fiir die Phy-
siologic. Z .pliysiol.Cliem., 26, 60-87.
Fitzgerald, G.F., Chance or vitalism. Nature, 58, 545 ;
59, 76.
Japp, F.R., Stereochemistry and vitalism. Presidential
address. Nature, 58, 452.
Kipping, F.S. and W.J. Pope, Stereochemistry and
vitalism. Nature, 59, 53.
Pearson, K., Asymmetrv and vitalism. Nature, 58, 495 ;
also 5.9, 30, 124.
136 GENERAL BIBLIOGRAPHY
Pere, a., Fermentation lactique des corps siicres par
le Coli-bacille. Ami.Inst. Pasteur, 12, 63-72.
PoTTEviN, A., Contribution a Petude de la fermenta-
tion lactique. Ann.Inst.Pasteur, 12, 49-62.
Spencee, H., Stereochemistry and vitalism. Nature,
58, 592.
Steoxg, W.M., Stereochemistry and vitalism. Nature,
59, 53.
1900
Fischer, E., Spaltung- einig-er racemischer Amino-
sauren in die ojjtisch-aktiven Komponenten. Ber.chrm.
Ges., 32, 3638-36-t6.
MoRiSHiMA, K., tjber das Vorkonnnen der Milchsaure
im tierischen Org-anismus mit Beriicksichtigung der Ar-
senvergiftung-. Aycli.exp.Path.Pharma'k., J/S, 217-244.
Neuberg, C, Uber die Harnpentose, ein optisch-inak-
tives, natiirlich vorkommendes Kohlenhydrat. Ber.chem.
Ges., 33, 2243-2254.
Walden, p., Fiinfundzwanzig Jahre stereochemischer
Forschung. N at ur wis s. Rundschau, 15, 145-148.
1901
OsHiMA^ K. AND B. Tollens, Ubcr das Nori aus Japan.
Ber.chem.Gcs., 3',, 1422-1424.
1902
Neuberg, C. and J. Wohlgemuth, Uber das Ver-
halten stereoisomerer Substanzen im Tierkorper. I.
Uber das Schicksal der drei Arabinosen im Kaninchen-
leibe. Z .physiol.Cltem., 35, 41-69.
1903
CusHisTY, A.E., Atropine and the hyoscy amines — a study
of the action of optical isomers. J.Physiol., 30, 176-194.
McKenzie, a. and a. Harden, The biological method
for resolving inactive acids into their optically active
components. J.Chem.Soc, S3, 424-438.
GEXERAL BIBIAOGRAPHY 137
Neuberg, C. and p. Mayee, tJber das Verhalten ste-
reoisomeror Substaiizoii im Tierkorper. II. Uber das
Schicksal der drei Mannosen im Kaninchenleibe. Z.phy-
siol.Chem., 37, 530-544.
1904
Byk, a., Zur Frage der Spaltbarkeit von Kacemver-
biiidiiiigen durch zirkularpolarisiertes Licht, ein Beitrag
zur priniareu Entstehung optisch-aktiver Substanz.
Z.physikal.Che'm., ',9, 641-687.
CoNDELLi, S., tJber die Spaltung der racemisclieii Wein-
saure durcli Aspergillus niger. Chem.Zhl., 2, 824.
Dakin, H.D., Tlie fractional hydrolysis of optically
inactive esters by lipase. J.Pliysiol., 32, 199-206.
Marckwald, W., Uber asymmetrische Synthese. Ber.
cliem.Ges., 31, 349-354.
JMcKenzie, a., Studies on asynnnetric synthesis.
J.Chem.Soc, 83, 1249-1262.
Pictet, a. and a. Eotschy^ Synthese des Nieotins.
Ber.cliem.Ges., 31, 1225-1235.
1905
CusHNY, A.R. AND A. Peebles, Tlic action of optical
isomers. II. Hyoscines. J.Physiol., 32, 501-510.
ScHULZE, E. AND E. WiNTERSTEiN, Ubcr das spezi-
fische Drehungsvermogen einiger aus Pflanzen darge-
stellten Tyrosinpraparate. Z.physiol.Chem., Ji5, 79-83.
Walden, p., Uber das Drehungsvermogen optisch-
aktiver Korper. Ber.cliem.Ges., 38, 345-409.
Wohlgemuth, J., Uber das Verhalten stereoisomerer
Substanzen im tierischen Organismus. Die inaktiven
Monoaminosauren. Ber.cliem.Ges., 38, 2064-2065.
1906
Abderhalden^ E. and F. Samuely, Der Abbau des Leu-
cins und des Leucyl-leucins im Organismus des Hundes.
Z.physiol.Chem., )7, 346-353.
138 GENERAL BIBLIOGRAPHY
Ehrlich, F., tJber eine Methode zur Spaltung racemi-
scher Aminosaiiren mittels Hefe. Biochem.Z., 1, 8-31.
Engler, C, Zur Frage der Entsteliuiig des Erdols.
CJiem.Zeitwig, 30, 711-714.
Mayer, P., Uber die Spaltung der lipoiden Substan-
zen durcli Lipase und liber die optischen Antipoden des
natiirlichen Lecithins. Biochem.Z., 1, 39-52.
Meyerhoffer, W., Gleicligewiclite der Stereomeren.
Neuberg, C, tJbor die Entstehung optisch-aktiver Fett-
sauren in der Natur. Biochem.Z., /, 368-379.
1907
Abderhalden, E. and a. Schittexhelm, Studien iiber
den Abbau racemischer Aminosauren im Organismus des
Hundes unter verscliiedenen Bedingungen. Z.physiol.
Chem., 51, 323-333.
Fischer, E., Vorkommeu von 1-Serin in der Seide.
Ber.chem.Ges., J/O, 1501-1505.
Fischer, E. and K. Raske, Verwandlung des 1-Seriiis
in d-Alanin. Ber.chem.Ges., JjO, 3717-3724.
Neuberg, C, Die Entstehung des Erdols, Bildung von
optisch-aktiver Naphtha aus drehenden Fettsauren.
Biochem.Z., 7, 199-212.
Neuberg, C. an^d E. Rosenberg, Verwandlung von op-
tisch-inaktiven Triolein in ein optisch-aktives Glycerid
und eine optisch-aktive Saure. Biochem.Z., 7, 191-198.
Stoklasa, J., Uber die glykolytischen Enzyme im Pflan-
zenorganismus. Z.physiol. Chem., 50, 303-360; also 51,
156-157.
1908
Bredig, G. and K. Fajans, Zur Stereochemie der Kata-
lyse. Ber.chem.Ges., J/l, 752-763.
CusHNY, A.R., The action of optical isomers. III.
Adrenalin. J.Physiol., 37, 130-138.
Ehrlich, F., Uber die Spaltung racemischer Amino-
sauren mittels Hefe. 11. Biochem.Z., 8, 438-466.
GENERAL BIBLIOGRAPHY 139
Ehrlich, F. axd a. Wende-l, Zur Kenntnis cler Leucin-
fraktion des Eiweisses. Biocliem.Z., N, 399-437.
RosENTHALEE, L., Duvcli Eiizyiiie bewirkte asymiiie-
trisclie Syntlieson. Biochem.Z., 1'/, 238-253; also 17, 257-
269, 1909.
VAN Ekenstein, W.A, and S. Blanksma, Beitrag zur
Kenntnis der Zucker. Chem.Zhl., 119.
1909
CusHNY, A.R., Eurtlier note on adrenalin isomers.
J.PlujsioL, 38, 259-262.
Neubeeg, C, Yerlialten von racemiselier Glutaminsaure
bei der Faulnis. Biochem.Z., 18, 431-431.
Neuberg, C. axd L. Karczag, Verhalten von dl-Valin
bei der Faulnis. Biochew.Z., IS, 435-439.
Walker, W. axd V. Krieble, The amygdalins. J.Chem.
Soc, 0.'), 1437-1449.
1910
Bartelt, K., Terpene und Camplier. Ahderh.Bioch.
Eandlex., 7, 266-538.
Fajans, K., Uber die stereochemisclie Spezifizitat der
Katalysatoren ( Optisclie Aktivierung durch asymme-
trische Katalyse). Z.physikal.Chem., 73, 25-96.
Karczag, L., Beitriige zur physiologischen Wirkung der
Weinsauren. Z.Blol., o3, 218-231.
Leger, E., Sur I'aloinose ou sucre d'aloine. C.R.Acad.
ScL, loO, 983-986.
, Sur I'aloinose cristallise; son identite avec
I'arabinose -d. C.R.Acad.Sci., 150, 1695-1697.
Pringsheim, H., Notiz fiber das Vorkommen von
Rechts-Asparagin in der Natur. Z .physiol.Chem., Go, 89-
95.
, Studien iiber die Spaltung racemi-
selier Aminosauren durcli Pilze. Z .physiol.Chem., Go, 96-
109.
140 GENERAL BIBLIOGRAPHY
1911
CuEEiE, J.N., The study of the optical forms of lactic
acid produced by pure cultures of Bacillus hulgaricus.
J.BioLChem., 10, 201-211.
Neuberg, C. and S. Saneyoshi, tJber das Verhalten der
stereoisomeren Weinsauren im Organismus des Hundes.
B loch em. Z., M], 32-36.
Thiele, a., Oxydicarbonsauren. Ahderli.Biocli. Hand-
lex., 1, 1149-1182.
1912
Bredig, G. and p. Fiske, Durcli Katalysatoren be-
wirkte asymnietrische Synthese. Biochem.Z., .'/(i, 7-23.
Embden, G., Baldes, K. and E. Schmitz, Uber den
Chemisnius der Milchsaurebildung' aus Traubonzucker im
Tierkorper. Biochem.Z., 'i'), 108-133.
Krieble, v., The amygdalins and their inter-relations
with emulsin. J.Ani.Chew.Soc, 3'/, 71(1-735.
1913
Krieble, v., Asynnnetric synthesis through the action
of oxynitrilases. J.Am.Chcm.Soc., 35, 1643-1647.
Net'berg, C, Weitere Untersuchungen liber die bio-
chemische Umwandlung von Methylglyoxal in IMilch-
saure nebst Bemerkungen iiber die Entstehung der ver-
schiedenen Milchsauren in der Natur. Biochem.Z ., 51,
484-508.
Study, E., Die Begritfe Links, Kechts, Windungssinn
und Drehungssinn. Arch.Mafh. mid Physih, 21, 193-226.
1914
Ehrlich, p., Uber asynmietrische und symmetrische
Einwirkung von Hefe auf Racemverbindungen natiirlich
vorkommender Aminosauren. Biochem.Z., 1)3, 379-401.
1916
Peingsheim, H. and E., Die Chemotaxis von Bakterien
gegen optisch-aktive Aminosauren. Z .physiol.Cliem., 97,
176-190.
(!i:\i:ir\i. iniUjiodRAPHY 141
1918
Clough, G.W., The relationship between the optical ro-
tatory powers and the relative contiourations of optically
active componnds. .1 .(liem.Soc, //.>, 526-554.
1919
CusHNY, A.E., On optical isomers. V. The tropeines.
J.PlicumacoL, i.>, 71-93.
Jaeger, F.M., Untersnchnngen iiber das Pasteursche
Prinzip. Bpc.frav.chim.Pays-Bas, N, 171-314.
McKexzie, a. and J. Wood, The isomeric tropic acids.
J.Chem.Soc, 115, 828-840.
Oela- Jensen, The lactic acid bacteria. Copenhagen.
1920
Faeber, E., F. Xord and C. Xeubp:rg, Die phytochem-
ische Reduktion des Acetols zii optisch-aktiven Propylen-
glykol. Biochem.Z., 112, 313-323.
Hess, K. and W. Weltzien, Uber die Fahigkeit der
Pflanze, optische Antipoden aufzubauen. Ber.chem.Gcs.,
JJ, 119-129; 1375-1378.
Pringsheim, H., Uber das Vorkonnnen optischer Anti-
poden in der Xatur. Ber.chem.Ges., 53, 1372-1374.
1922
Gersbach, a., Uber die Wendigkeit der Kolonieauslau-
fer des Bacillus mycoides (''Isomeric" bei Bakterien).
Zhl.BaU., 88, 97-103.
KoTAKE, J. AND M. Okagawa, Ubcr den Einfluss des op-
tischen Drehungsvermogens auf die Zellpermeabilitat.
J.BiocJiem. {Tokyo), 1, 159-164.
NoRDEFELDT, E., Ubcr die asymmetrische Wirkung des
Emulsins bei der Benzoxynitril-Synthese. Biochem.Z.,
131, 390-410.
192
Q
Braun, J. AND W. Kaiser, Geruch und molekulare/^Gil C
Asymmetric. Ber.chem.Ges., 56, 2268-2274. /'^'^ -'oi"
^^,'^
LI5R
142 GENERAL BIBLIOGRAPHY
Freudenberg, K., F. Brauxs and K. Siegel, Die Konfig-
uration der Manclelsiiure mid anderer d-Oxysauren. Ber.
chem.Gcs., 56, 193-200.
Jaeger, F.M., Sur le pouvoir rotatoire des composes
cliimiques en rapport avec leur configuration moleculaire
et la theorie des atoms dits asymetriques (Conference).
Bull.soc.chini. France, S3, 853-889.
WoHL, A. AND K. Freudenberg, Die Bezeichnung ster-
isclier Reilien. Ber.chem.Ges., 56, 309-313.
1924
Freudenberg, K. and F. Rhino, Die Konfiguration des
Alanins. Ber.chem.Ges., 51', 1547-1557.
1925
Bragg, W. and R. Gibbs, The structure of a and |3
quartz. Proc.Roy.Soc.A., 109, 405-427.
Braun, J. and W. Teuffert, Geruch und molekulare
Asymmetric. II. Ber.chem.Ges., 58, 2210-2215.
Byk, a., Zur Synthese der molekularen Asymmetric.
Naturwiss., 13, 17-21.
Langenbeck, W., Der Abbau des 1-Histidins zur 1-As-
paragin-saurc. Ber.chem.Ges., 58, 227-229.
Neuberg, C. and G. Gorr, tJber den Mechanismus der
Milchsaurebildung bei Bakterien. Biochem.Z ., 166, 482-
487.
Okagawa, M., tJber den Einfluss des optischen Dre-
hungsvermogens auf die Zellpermeabilitat. Z.physiol.
Chem., 1.1,3, 211-217.
1926
Binder-Kotrba, G., Die Umwandluug von Phenylgly-
oxal in Mandelsaure durcli die Ketonaldehydmutase grii-
ner Pflanzen. Biochem.Z., 17 '/, 443-447.
CusHNY, A.R., Biological relations of optically isomeric
substances. Baltimore.
Karrer, p. and M. Ehrenstein, Zur Kenntnis einiger
natiirliclien Aminosauren. Helvef.Chim.Acta, 9, 323-331.
GENERAL BIBLIOGRAPHY 143
Mayek, p., Zur Biochemie des Asymmetrieproblems.
BiocJiew.Z., /r//, 420-424.
Neuberg, C and E, Simox, Beitrage zur Biochemie des
Asymmetrieproljlenis ( Asymmetrisclie Dismutation) .
B'wchem.Z., 179, 443-450.
PEDERsoisr, C, W. Peterson and E. Fred, The forms of
lactic acid produced by pure and mixed cultures of bac-
teria. J.BiolChcm., 68, 151-164.
1927
Neuberg, C. and M. Kobel, fiber die Bildung von reiner
d(-) Milchsaure durch frische Hefen und Trockenhefe,
sowie von dl-Milchsaure durch Hefenmazerationssaft.
Biochem.Z., 182, 470-487.
Neuberg, C. and E. Simon, tjber die quantitative Dismu-
tation des Methylglyoxals zu Milchsaure durch den Bacil-
lus Dclbriicki, sowie durch das Bacterium lactis aerogenes
nebst Versuchen iiber das abweichende stereochemische
Verhalten des Phenjdglyoxals bei dieser Reaktion. Bio-
chem.Z., 186, 331-336.
1928
Neuberg, C. and E. Simon, Weitere Untersuchungen
iiber die stereochemische Spezifitat der Ketonaldehydmu-
tase. Biochem.Z., 200, 468-472.
1929
Bendrat, M., Zur Physiologie der organischen Sauren
in griinen Pflanzen. VI. Ein Beitrag zur Kenntnis des
Saurestotfwechsels sukkulenter Pflanzen. Planta, 7, 508-
584.
Boycott, A., C. Diver, S. Hardy and F. Turner, The in-
heritance of sinistrality in Limnaea peregra. Proc.Rog.
SocB., lO.'i, 152.
Fitting, H., Uber die Auslosung von Plasma stromung
durch optisch-aktive Aminosauren. Jahrh.Bot., 70, 1-25.
Hayashi, S., Experimentelle Untersuchungen iiber die
sterischen Verhaltnisse bei der Dismutation von Phenvl-
144 GENERAL BIBLIOGRAPHY
glyoxalhydrat durcli verschiedene Bakterien. Biochem.Z.
206, 223-'227.
KuHN, W. AND E. Braun, Pliotocliemisclie Erzeuguiig
optisch-aktiver Stoffe. Nafunmss., 11, 227-228.
Macht, D., Pharmacological synergism of stereoisom-
ers. Proc.Nat.Acad.Sci.U.S.A., 15, 63-70.
Ruhland, W. and K. Wetzel, Zur Physiologic der or-
ganischen Sauren in griinen Pflanzen. V. Weitere Un-
tersuchungen an Rheum hyhrldum. Plant a, 7, 503-507.
WiDMANN, E., Quantitative Umwandlung von Methyl-
glyoxal in reine d(-) Milchsaure durch das Bacterium
fluorescens. Biochem.Z., 2 16, 475-478.
1930
Bamann, E. AND P. Laeverenz, Ubcr den Einfluss von
optisch-aktiven Fremdstoifen auf die Kontigurations-
Spezifitat der Leber-Esterase verschiedener Tiere. Ber.
chem.Ges., 63, 394-404.
Erlenmeyer, H., Studieu zu einer dynamischen Stereo-
chemie. Helvet.Chim.Acta, IS, 731-747.
KuHN, W. AND E. Knopf, Pliotocliemisclie Erzeugung
optisch-aktiver Stoffe. Naturwiss., IS, 183.
1932
Bredig, G. AND F. Gerstner, Asymmetrische Katalyse
mit organischer Faser. Biochem.Z., 230, 414-429.
Bredig, G. and M. Minaeff, Asvmmetrische Svnthese
durch Katalysatoren. II. Biochem.Z., 2',9, 241-244.
KuHN, W. AND K. Freudenberg, Dreliuug der Polari-
sationsebene des Lichtes. Jahrh.Chem.Physik, N, 1-142.
Ludwig, W., Das Eechts-Links-Problem. Springer,
Berlin.
McKenzie, a., tJber asymmetrische Synthese und ilire
Entwicklung. Angeiv.CUem., J/o, 59-65.
Mills, W.H., Some aspects of stereochemistry. J.Soc.
Chem.Ind., 51, 750-759.
OuDiN, A., Sur les variations considerables que pre-
sente, d'un arbre a 1 'autre, le pouvoir rotatoire de I'es-
GEM:/f\L nffUJOCLW/'HY 145
sence de terebontliiiio du pin iiiaritiinc (Pinus pinaster
Sol.) et la fixite relative de ce pouvoir rotatoirc pour un
arbre donne. C.R.Acad.Sci., 193, 185-186.
ScHWARZE, P., Ein Beitrag' zur Kenntiiis des Siiure.sloff-
wechsels niclitsukkulenter Pflanzen. Planta, 18, 168-210.
Tatum, E., W. Peterson and E. Fred, Effect of asso-
ciated growth on forms of lactic acid produced by cer-
tain bacteria. BiocJiemJ., 26, 846-852.
1933
Embden, G., H. Deuticke and G. Kraft, Uber die in-
termediaren Vorgange bei der Glykolyse in der Muskula-
tur. KUu.Woclienschr., 12, 213-215.
Krebs, H. a., Untersuchungen liber den Stoff'weclisel
der Aminosauren im Tierkorper. Z.physiol.Chem., 211 ,
191-227.
Orechoff, a., ]\[. Kabinowitch and R. Konowalowa,
tJber Sopliora-Alkaloide. Ber.chem.Ges., 66, 621-625.
Ritchie, P., Asymmetric synthesis and asymmetric in-
duction. Oxford.
Rona, p. and R. Ammon, Die stereochemische Spezi-
fitiJt der Esterasen und die synthetisierende Wirkung der
esterspaltenden Fermente. Ergehn.Enzymforsch.,2, 50-73.
Stoll, a. and E. Wiedemann, Die optische Aktivitat
des Chlorophylls. Helvet.Chim.Acta, 16, 307-314.
1934
Boys, S.F., Optical rotatory power. Parts I and II.
Proc.Roij.Soc.A., 1.',.',, 655-692.
Du Vigneaud, v., H. Craft and H. Loring, The oxida-
tion of the stereoisomeres of cystine in the animal body.
JMiolChem., lOJ,,- 81-89.
Edlbacher, S. and M. Neber, Zur Kenntuis des inter-
mediaren Stoffwechsels des Histidins. Z.physiol.Chcm.,
22',, 261-272.
Freudenberg, K. and F. Nikolai, Die Konfiguration
des Ephedrins. Lieh.Ann.Chem., 510, 223-230.
146 GENERAL BIBLIOGRAPHY
KoLTZoFF, N.K., Genetics and physiology of develop-
ment. Biol.J. (Rtissian), 3, 420-456.
Needham, J., Morphology and biochemistry. Nature,
IS',, 275-276.
RicHTER, G. AND R. DossER, Ubcr die Adsorption op-
tischer Isomeren am Nervengewebe. Biochem.Z., 268,
399-405.
Vernadsky, W., Problems of biochemistry. I. The im-
portance of biogeochemistry for the knowledge of the
biosphere. Leningrad; also IV. Concerning dextrality
and sinistrality. Moscow, 1940 (in Russian).
1935
Abderhalden", E. and E. Tetzner, Beitrag zur Kennt-
nis des Verhaltens racemischer Aminosauren im tier-
ischen Organismus. Z .physiol.Chem., 232, 79-86.
Ackermann, D., Die stickstoffhaltigen Endprodukte
der niederen Tierwelt. Uspehi Sovremennoi Biologii
(Moscow) //, 5-10.
Ammon, R., Die Bedeutnng der Stereochemie in der
Physiologic, speziell in der Fermentforschung. Erg.Phy-
sioL, 37, 366-405.
Bernheim, F., The oxidation of 1- and dl-tyrosine by
the livers and kidneys of various animals. J.Biol.Chem.,
Ill, 217-224.
Brockmann, H. and H. Roth, tJber spiegelbildliche
Naturfarbstoffe. Naturwiss., 23, 246.
KiscH, B., Beeinflussung des Gewebsstoffwechsels
durch optisch-aktive Aminosauren. Biocliem.Z., 280, 41-
54.
Krebs, H.A., Metabolism of amino acids. TIL Deamin-
ation of amino acids. Bioch.J., 29, 1620-1644.
LowRY, T.M., Optical rotatory power. Longmans,
Green and Co. London.
Mathieu, J. p., La synthese asymetrique. Actualites
Scientifiques, No. 209. Paris.
GENERAL BIBLIOGRAPHY 147
1936
Gause, G.F.,- Raumaufban des Protoplasmas. Ergehn.
Biol, 13, 54-92.
Krebs, H.A., Metabolism of amino acids and related
substances. Aiui.Rev.Blocliem., J, 247-270.
KuHN, W., Optische Spezifitat von Enzymen. Ergehn.
Enzymforscli, 5, 1-48.
, Katalytisclie Erzeiigung optiscli aktiver
Stoffe mid chemisclie Notwendigkeit eines einseitigen
Ablaufs biochemisclier Vorgange. Angew.Chem., J/O, 215-
219.
Langenbeck, W. and G. Triem, Zur Theorie der Er-
haltung iind Enstehung optisclier Aktivitat in der Natur.
Z.'phgsikal.Chem., 177, 401-408.
LuDwiG, W., Bestimmmig und Vererbung der Asym-
metrieform. Verh.Zool.Ges., 38, 21-73.
McKenzie, a., Asymmetric synthesis. Ergehn.Enzym-
forsch, J, 49-78.
Neber, M., tJber die oxydative Desaminierung von Am-
inosauren. Z.phgsiol.Chcm., 2.'i0, 59-69.
ScHOEN, M., Problemes d'asymetrie dans les processus
biochimiques. Actualites Scientifiques, No. 444, Paris.
Tatum, E., W. Peterson and E. Fred, Enzymic racem-
isation of optically active lactic acid. BiochemJ., SO,
1892-1897.
Webster, M. and F. Bernheim, Oxidation of amino
acids by Bacillus pyocyaneus. J.Biol.CJiem., IIJ/, 265-
271.
1937
Bennet-Clark, T., Organic acids in plants. Ann.Rev.
Biochem., 6, 579-594.
Branke, J. AND J. Parishev, Plauts of the Maritime
Region and their Volatile Oils. Vladivostok (in Rus-
sian).
Bruckner, V. and G. Ivanovics, tJber das natiirliche
Vorkommen und liber eine einfache biologische Gewin-
148 GENERAL BIBLIOGRAPHY
iiungsart der d(-) Glutaminsaure. Z.phi/slol.Chem., 2//7,
281-284.
Conrad, R. and C. Berg, The optical inversion of d-liis-
tidine in the animal body. J.Biol.Chem., 117, 351-363.
Du ViGNEAUD, V. AND 0. Irish, The role of the acetyl
derivative as an intermediary stage in the biological
synthesis of amino acids from keto acids. J.Biol.Chem.,
122, 349-370.
FiNDLAY, A., Use of the name 'Racemic acid." Nature,
I'lO, 22.
Katagiri, H. and K. Kitahara, Racemiase, an en-
zyme which catalyses racemization of lactic acids. Bioch.J.
31, 909-914.
KoPELOFF, L. AND N., Optical activity of lactic acid pro-
duced by L.acidopliilus and L.hulgaricus. J.Bact., 33,
331-334.
KoTAKE, Y. AND S. GoTO, Studicn iiber den interme-
diaren Stoffwechsel des Tryptophans. XXVII. Uber die
Konfigurationsanderung des d-Tryptophans im Tierkor-
per. Z.physiol.Chem., 2Jj8, 41-56.
Levene, p. and S. Mardashew, Configurational rela-
tionships of the aliphatic and aromatic amino acids.
J.Biol.Chem., Ill, 179-182.
Levene, P., A. Rothen and M. Kuna, The mechanism
of the reaction of substitution and Walden inversion.
J.Biol.Chem., 120, 111-191.
Menshikov, G., The structure of heliotridane and syn-
thesis of dl-heliotridane. Bull.Acad.Sci.U .S.8.B., Ser.
chem. (Russian) 5, 1035-1048.
Needham, J. AND H. Lehmann, Intermediary carbohy-
drate metabolism in embryonic life. VIII. Glyceralde-
hyde and glucolysis. Biochem.J., 31, 1913-1925.
Pfeiffer, p. AND W. Christeleit, Komplex chemische
Methode zur relativen Konfigurationsbestimmung der
natiirlichen a-Aminosauren. Z .physiol.Chcm., 2'i-), 197-
210.
Rainey, R.C, Absolute configuration of the naturally
occurring a-aminoacids. Nature, I'lO, 150.
aEM:h'.\L liiiiLioiiuArin 149
Spiees, C, Zur Frage der Enstehuiig optischer Aktivi-
tat in der Natur. Naturwiss., 2.7, 457.
ToMiYAsu, G., Weiteres iiber die Existenz der Carboli-
gase, unter besonderer Beriicksichtigung der optischeii
Eigenschaft des Reaktionsproduktes. Biochem.Z ., 292,
234-240.
Tromsdorfj-, W., Das Verhaltiiis der Aiizahl der Links-
quarze zu den Rechtsquarzen in einer grosseren Menge
von Qiiarzkristallen. Neue Jahrh.Mineralogic, 72, 464-
495.
1938
Gause, G.F., The problem of asymmetry of protoplasm.
Acta Biotheoretica, .), 1-24.
Gause, G. F. and N. p. Smaragdova, An analysis of
growth of the yeast Torula ut'ilis on optically isomeric
leucines and valines. B'loJJ. (Russian), 7', 399-411.
, Some features in
the evolution of integuments in fresh-water animals ana-
lysed by killing action of optically isomeric organic acids.
BiolJ., (Russian), 7, 937-950.
Gause, G.F., N.P. Smaragdova and W.W. Alpatov, On
the action of optically isomeric cinchonines upon various
functions of a cell containing and non-containing symbio-
tic zoochlorellae (Experiments on Paramecium caudatum
and Paramecium hursaria). Biol.J., (Russian), 7, 763-
776.
Mendel, B., F. Strelitz and D. Mxjndell, L-glyceric al-
dehyde and tumor metabolism. Science, 88, 149-150.
Zvetkoff, B.N., Dextral and sinistral forms of the
mollusc Fruficicola lantsi Lndli. and their geographic dis-
tribution. BuU.Soc.Nat.Moscou, Ser.BioL, 'p. 414-419.
1939
Arnow, E. and J. Opsahl, The configuration of the
glutamic acid of adenocarcinoma protein. Science, 90,
257-258.
150 GENERAL BIBLIOGRAPHY
Bayeele, H., Uber die Verdaulichkeit von Tumorge-
webe durcli normale Enzyme. Biochem.Z., M3, 251-259.
Chibxall, a., M. Eees, G. Tristram, E. Williams and
E. BoYLAND, The glutamic acid of tumour proteins. Na-
ture, lJ,'f, 71-72.
DiTTMAR, C, Tiber das Vorkommen von d(-)Glutamin-
saure in Tumorproteinen. Z.Krehsforscli., .'/D, 397-398.
Du ViGNEAUD, v., M. CoHiq^, G. Brown, 0. Irish, R.
SCHOENHEIMER AND D. RlTTENBEEG, A Study of tlie luver-
sion of d-phenylamino-butyric acid. J .Biol. Ch em., ISlj
273-296.
Gause, G.F., Some physiological properties of dextral
and of sinistral forms in Bacillus mycoides Fliigge. Biol.
Bull., 76, 448-465.
Gause, G.F. and N.P. Smaragdova, Temperature char-
acteristics of toxic action of optically isomeric organic
acids. Bull.Biol.Med.Exper.U.S.8.B., 7, 105-107.
, Effect of isomeric
tartaric acids upon the metabolism of lower organisms
and vertebrates. Biai.Biol.Med.Exper.U.S.S.B., 7, 108-
110.
, On the killing ac-
tion of optically isomeric nicotines in relation to prob-
lems of evolution of the nervous system in animals.
PhysioLZool, 12, 238-255.
Graff, S., Glutamic acid in malignant tumors. J.Biol.
Cheni., 130, 13-17.
KiESEL, A., E. Efimochkina and J. Rall, On the pos-
sible relation between the asymmetry of the organism and
optical activity of the substances it is composed of. I.
Amino-acids from the conchiolin of shells with right and
left direction of the whorl. C.B.Acad.Sci.U.S.S.R., 25,
481-483.
KoGL, F., Zur Atiologie der Tumoren. Z.Krehsforscli.,
J,9, 291-311.
KoGL, F. and H. Erxleben, Zur Stereochemie der Pro-
teine von Myomen und anderen Geschwiilsten. Z.physiol.
Chem.,261,'1^^-111.
ge\i:ra l liiBLiodir \ /'// v 151
, The jiiutaiiiic acid of tumor
proteins. Xafure, ///'/, 111.
, Zur Isolierung der Glutam-
iiisaure aus Tumorproteinen. Naturiviss., 27, 486.
, Zur Atiologie der malignen
Tumoren. Z.pliysloLCliem., 258, 57-95.
KoGL, F., H. Erxlebei^ and A. AivKERMAisr, Isolierung
der Glutaminsaure aus Tumorproteinen. Z .physiol.Chem.,
261, 141-153.
Lemmleiist, G., On the number of sinistral and dextral
cristals of quartz in a single deposit. Trav.Lab.Biogeo-
chimique de VAcad.Sci.U.S.SM. (Russian), J, 225-237.
Waldschmidt-Leitz, E. and K. Mayer, Uber sterische
Auslese durcli Peptidasen in normalen und carcinoma-
tosen Seren. Z .physiol.Chem., 262, IV- VI.
White, J. and F. White, On the nature of the glutamic
acid of malignant tumor tissue. J.Biol.Chem., 130, 435-
436.
1940
Abderhalden, E. and G. Caesar, Uber das Verhalten
von Dipeptiden, in denen Aminosauren der d-Eeihe ent-
halten sind, bei der Einwirkung von Presssaften aus Ge-
weben und von Blutserum. Fermentforsch., 16, 299-308.
Arnow, E. and J. Opsahl, The attempted characteriza-
tion of malignant tissue protein with d-amino acid oxi-
dase. Science, 91, 431-432.
Bayerle, H. and F. Podloucky, Zur Frage des Vorkom-
meiis von sterisch auslesenden Enzymen im carcinoma-
tosen Organismus. Z. physiol.Chem., 26.), 189-197.
, Zur Frage des Vorkom-
mens von d-Peptidasen in normalen und carcinomatosen
Organen. Biochem.Z., SOJ^, 259-265.
Chibnall, a., M. Rees, E. Williams and E. Boyland,
The glutamic acid of normal and malignant tissue pro-
teins. Biochem.J., 31,, 285-300.
, Glutamic
acid of proteins. Nature, 11,-5, 311-312.
152 GENERAL BIBLIOGRAPHY
Edlbachee, S., M. Simon and M. Becker, tJber den Ab-
bau des d-Histidins beim Meerschweinchen. Naturwiss,,
28, 411-412.
Erxlebex, H. Axi) H. Herkex, Beitrage zur stereochem-
ischen Analyse von Proteinen des Lymphog-ranuloms und
anderer patliologischen Gewebe. Z.physiol.Chem., 26Jt,
240-250.
Frutox, J., G. Irving and M. Bergmann, The specifity
of proteolytic enzymes from tumors. J .Biol.Chem., 132,
465-466.
Gause, G. F. and N. p. Smaragdova, The decrease in
weight and mortality in dextral and sinistral individuals
of the snail Fruticicola lantzi. Am. Naturalist, 77/.
Graff, S., D. Rittenberg and G. Foster, The glutamic
acid of malignant tumors. J .B'wl.Chem., 133, 745-752.
Herken, H. and H. Erxleben, tJber den Nachweis von
d-Peptidasen mit Hilfe von d-Aminosiiureoxydase. Z.
pliysiol.Cliem., 2i)'i, 251-253.
Johnson, J., A comparison of the optical forms of glu-
tamic acid from normal and cancerous tissue. J.Biol.
Chem., 132, 781-782.
Kogl, F. and H. Erxleben, Uber das Verhalten nor-
maler Verdauungsfermente gegeniiber Tumorproteinen.
Z.physiol.Chem., 26',, 108-122.
, Methodisches zur Isolier-
ung der Glutaminsaure-Antipoden. Z .physiol.Chem., 26.^,
198-219.
Kogl, F., H. Erxleben and H. Herken, Weitere Bei-
trage zur stereochemischen Analyse von Tumorproteinen.
Z.physiol.Chem., 263, 107-124.
Kogl, F., H. Herken and H. Erxleben, Uber die Son-
derstellung der d-Glutaminsaure bei Versuchen mit
d-Aminosaureoxydase. Z .physiol.Chem., 261,, 220-239.
Konikova, a., Glutamic acid of proteins. Nature, I'fO,
312.
Lipmann, F., 0. Behrens, E. Kabit and D. Burk, The
determination of the total d-amino acid content of human
GENERAL BIBLIOGRAPHY 153
tumors and normal tissues by means of d-amino acid
oxidase. Science, 91, 21-23.
Ratner, S., R. Schoenheimer and D. Rittenberg, The
metabolism and inversion of d( + )leucine studied with
two isotopes. J.Biol.Chew., IS), 653-663.
Schramm, G. and H. Miller, Uber die Konfiguration
der ini Tabakmosaikvirus enthaltenen Aminosauron. Na-
turwiss., 28, 223-224.
Town, B., Glutamic acid of proteins. Nature, U/o, 312-
313.
Waldschmidt-Leitz, E. and R. Hatschek, Bemerkun-
gen zur Arbeit von Bayerle und Podloucky. Z.physiol.
Chem.,26',, 196-197.
Waldschmidt-Leitz, E., K. Mayer and K. Hatschek,
tjber das Auftreten von d-Peptidase im Serum als Ab-
wehrreaktion. Z.pltysiol.Chem., 26S, I-III.
SUBJECT INDEX
Abies, 30
Acanthis, 103, 117
Adonis, 29
Aelosoma, 118, 119, 120
Alkanna, 29
Amoeba, 116
Amomum, 29
Andropogon, 29
Anethion, 30
Apium, 30
Arenicola, 118
Asarium. 29
Aspergillus, 93
Asymmetric analysis, 99
induction, 37
state, transmission of, 35, 37
synthesis, 35, 36, 37, 38, 39, 41,
43, 45
Asymmetry, and cancer, 129
as a criterion of organic origin,
31
as a specific property of pro-
toplasm, 20
definition, 10
of primary constituents, 21
optical and geometrical, 11
origin of, 52, 53
Bacillus, 81, 82, 83, 84, 85, 86, 87,
88, 89, 91, 93, 95, 96
Bacterium, 62, 67, 68, 71
Balanus, 115
Barbados, 29
Bar asm a. 30
Biological series of isomers, 15, 17,
31
Blumea, 29
Canarium, 30
Caruvi, 30
Chaetogaster, 118, 119
Citrus, 30
Cladonema, 118
Clostridiuvi, 64
Correction, in maintaining optical
purity, 45, 50
C rot on, 8
Cyclops, 118
Dahlia, 22, 23
Dalyellia, 118
Daphnia. 118
Dextral and sinistral strains, physi-
ology of, 84, 89
Differential effect of optic isomers,
99, 120
Dissymmetric substances, produc-
tion from symmetric, 67
Dissymmetry, and configuration of
organic molecules, 14
and life, 19
and optical activity, 12
definition, 9
in organic nature, 19
in quartz, 13
morphological, 79
the loss of, 74
Drosophila, 118
Dryobalanops, 29
Erigeron, 30
Eucalyptus, 30
Euchlanis, 118
Euplotes, 118
Evasion, in maintaining optical
purity, 45
Foeniculum, 30
Fraxinns, 25
Fruticicola, 83, 84, 89, 90, 91, 93,
96
Fundulus, 115
Gammarus, 118
Genes, optical inversion of, 81
Helobdella, 118, 119
Hydra, 116, 118
Inversion, morphological, 79
morphological and optical, rela-
tion of. 91
optical, 15, 59, 61
Juniperus, 30
Ketonaldehydemutases, 67
Lacerta, 103, 117
Lactobacillus, 62, 65, 66, 68
Laurus, 26, 29, 30
155
156
SUBJECT IXDEX
Lavandula, 29
LeMstes, 105, 106, 109, 110, 117
Leptoplana, 118, 119
Leuciscns, 101, 102, 103, 104. 117
Leuconosioc, 65, 66
Limnaea, 79, 80
Limnodrilvfi, 118, 119, 120
Lindera. 30
Linens, 118
Hthospermnm, 29
Lupinus, 22, 23
Massoia, 30
Mechanism, of Langenbeck and
Triem, 46, 54, 56
of Krebs, 48, 54, 55, 56
of Kuhn, 54
Mentha, 30
Monodora, 30
Mucor, 71
Myristica, 30
Myrrha. 30
Myrtus, 30
2^ais, 118, 119, 120
Natural isomers, 28
Nervous system, evolution of, 116
Nicotiana, 25
Optical activity, and dissymmetry,
12
heredity and environment, 59
stability of, 42
Optical inversion, 15, 59, 61
of genes, 81
of the Walden type 73, 74
Optical isomers, and intermediate
pathways, 70
differential action of, 99, 120
mechanisms controlling produc-
tion, 67
mechanism of action, 100
production by catalysts, 68, 69
production by esterases, 68
velocity of formation, 45
Optical purity, advantages of, 51
and 'fixed internal milieu," 45
maintenance of, 43, 45
Organic acids, dual action of, 112
Palaemon, 117
Paramecium, 107, 108, 118, 124, 125,
128
"Pathways", and optical isomers,
71
Pelargonium, 25
Perinereis, 118, 119
Petroleum, origin and asymmerty,
31
Peumus, 30
Phaenocora, 118
Phycomyces, 94
Physiological mutations, 62, 72
Picea, 30
Pitivs, 26, 29, 30
Piper, 30
Pittosporum, 30
Platyjioeciliis, 106, 109, 110, 111
Polycelis, 118
Porphyra, 23
Primary constituents of proto-
plasm, asymmetry of, 21
exclusiveness of the asymmetry
sign, 27
impossibility of inverting, 59
replaceability of, 28
Principle, Curie's, 36, 43, 54
of fixed pathway, 74, 75
Pristina, 118, 119
Procerodes, 118, 119
Priimis, 26, 44
Pyrethrum, 29
Quartz, optical activity, 12, 13
Racemates, splitting up of, 41
Racemiase, 65, 66
Racemic mixture, definition, 10
Racemization, and ageing, 47
and culture medium, 64
and temperature, 66
Rana, 103, 110, 117
Relative configuration, 15, 30, 72
Rheum, 25
Rotifer, 118
Rubus, 25
Saccharomyces, 71
Saccocirrus, 118, 119
Sagitta, 118, 119
SUBJECT INDEX
157
Secondary constituents of proto-
plasm, asymmetry of, 24
inverting of, 61 -
non-exclusiveness of the asym-
metry-sign, 28
Sempervirum. 25
Solidago, 30
Sophora, 29
SjXirtium. 29
Spiral growth, theory of, 93
SpirostoDiioti, 118
mentor, 118
Stereo-autouomic substances, 43,
44. 55
Stereo-coefficient, 101, 116
Stylaria, 118
Synergism, of stereoisomers, 100
Tanacetum, 29
Temporary dissymetric sub-
stances, 74
Thuja, 29
Thy VI us, 30
Torula, 60, 91, 92, 110
Trapaeolum . 23
Valeriana, 26, 30
AValden inversion, 73, 74
Xanthoxyluni, 30
AUTHOR INDEX
Abderhalden, 48, 137. 138, 146, 151
Ackermann, 19, 146
Akkerman, 151
Alpatov, 107, 108, 124, 125, 126, 149
Amnion, 68, 69, 70, 145, 146
Arkwright, 86
Arnow, 131, 149, 151
Bacq, 122, 123
Baldes, 71. 140
Bamann, 70, 144
Barcioft, 45
Bartelt, 26, 29, 30, 139
Bartium, 53, 135
Bayerle, 150, 151
Bayliss, 38
Becker, 152
Behrens, 152
Belloni, 29
Bendrat, 25, 143
Bennet-Clark, 24, 147
Berg, 61, 148
Bergmann, 152
Bernard, 45, 52
Bernheim, 126, 146, 147
Binder-Kotrba, 68, 142
Biot, 32
Blanksma, 29, 139
Borsook, 40
Bosshard, 48, 134
Bouchardat, 29
Boycott, 79, 143
Boyland, 150, 151
Boys, 14, 145
Bozler, 124
Bragg, 13, 142
Branke, 29, 147
Braun, E., 53, 144
Braun, I., 141, 142
Brauns, 72, 73, 142
Bredig, 38, 43, 68, 69, 70, 138, 140,
144
Brion, 135
Brockmann, 29, 146
Brown. F., 120
Brown, H., 23, 36, 135
Brown, O., 150
Bruckner, 95, 147
Burk, 152
Burton, 123
Byk, 52, 53, 137, 142
Caesar, 151
Cannon, 121, 122
Castle, 94
Chabrie, 135
Chibnall, 130, 150, 151
Christeleit, 31, 72, 148
Clough, 31, 141
Cohn, 150
Condelli, 137
Conrad, 61, 148
Cotton, 52
Craft, 145
Crozier, 103
Curie, 35, 135
Currie, 61, 140
Cushny, 114, 115, 136, 137, 138, 139,
141, 142
Dakin, 137
Deuticke, 71, 145
Dittmar, 150
Diver, 79, 143
Dosser, 146
Du Vigneaud, 61, 145, 148, 150
Edlbacher, 126, 145, 152
Efimochkina, 93, 150
Ehrenstein, 31, 142
Ehrlich, 22, 60, 138, 139, 140
Ekenstein, 29, 139
Embden, 71, 140, 145
Engler, 31, 138
Erlenmeyer, 144
Errera, 11, 53, 135
Erxleben, 23, 129, 130, 131, 150,
151, 152
Ewing, 129
Faerber, 24, 141
Fajans, 38, 43, 68, 138, 139
Findlay, 10, 148
Fischer, 10, 11, 16, 21, 22, 30, 36,
51, 55, 59, 73, 134, 135, 136, 138
159
160
AUTHOR INDEX
Fiske, 70, 140
Fitting, 143
Fitzgerald, 53, 135
Foster, 152
Franlte, 120
Fred, 61, 62, 143, 145, 147
Fresnel, 12, 13, 15, 133
Freudenberg. 16, 17, 72,
144, 145
Fruton. 152
73, 142,
Gause. 60. 84, 85, 86, 87, 88, 89, 90,
91, 92. 99. 100, 101, 102, 104, 105,
106, 107, 108, 109, 110, 113, 114,
115, 117, 118, 122, 123, 124, 125,
126. 147, 149, 150, 152
Gay-Lussac, 10
Gersbach, 82, 141
Gerstner, 144
Gibbs, 142
Gintl, 25. 133
Glaser. 124
Gold, 120
Gorr, 62. 142
Goto. 148
Graff, 130, 150, 152
Greenwood, 116, 117
Haller, 29
Harden, 136
Hardy, 79, 143
Hastings, 83
Hatschek, 153
Hayashi, 68, 143
Herken, 152
Hess, 27, 141
Hoagland, 123
Holmes, 50
Humboldt, 31
Irish, 148. 150
Irving, 152
Ivanovics, 95, 147
Jaeger, 19. 141, 142
Japp. 10. 11, 135
John-Brooks, 94, 95
Johnson, 152
Jungfleisch, 11, 134
Kabit, 152
Kaiser, 141
Kalmus, 124
Karczag, 139
Karrer, 31. 72, 142
Katagiri, 26, 61, 63, 64, 65, 66, 148
Kayser, 62, 63, 64, 66, 135
Kiesel, 93, 150
Kipping, 53, 135
Kisch. 48. 49, 146
Kitahara, 26, 61, 63, 65, 66, 148
Knopf, 53. 144
Kobel. 71, 143
Kogl. 23. 129, 130, 131, 150, 151, 152
Koltzoff, 80, 112, 146
Konikowa, 152
Konowalowa, 29, 145
Kopeloff. 66, 148
Kotake, 141, 148
Kraft, 71. 145
Krebs. 48. 49, 50, 51, 126, 145, 146,
147
Krieble. 38. 44. 139. 140
Kuhn. 26. 39, 41, 42, 43, 44, 47, 50,
53, 55, 144, 147
Kuna, 73, 148
Laeverenz. 70, 144
Langenbeck, 31, 43, 46, 47. 53,
147
Langer, 83
Langley, 120
LeBel, 134
Lees, 29
Leger, 29, 139
Lehmann. 148
Lemery. 31
Lemmlein. 20, 151
Lepeschkin, 113
Levene, 31, 73, 148
Lewis, 82, 87
Lipmann, 131, 132, 152
Lippman, 22, 134
Loewi, 121
Lomonosoff, 31
Loring. 145
Lowry, 12, 13, 15, 16, 19, 20, 146
Ludwig, 79, 82, 144, 147
Macht, 100, 144
Marckwald, 37, 137
142,
Al THOR lyOEX
161
Mardaschew, 31, 14S
Mathieu, 146
Mayer. K., 132, 151, 153
Mayer. P., 23, 68, 137, 138, 143
McKenzie, 27, 37, 136, 137, 141. 144,
147
Mendel, 149
Menschikov. 27, 148
Metalnikoff, 124
Meyerhol'fer, 138
Mills, 51. 53, 73, 144
Minaeff, 43, 70, 144
Morishima, 26, 136
Morris, 23, 36, 135
Muller, 153
Mundell, 149
Nachmanson, 122
Nay lor, 24
Neber. 48, 126, 145, 147
Needham, 80, 146
Nencki, 61. 135
Neuberg. 23, 24. 32, 62. 67, 68. 71,
74, 136. 137, 138, 139, 140, 141,
142, 143
Nikolai, 72, 145
Nord. 24. 141
Nordefeldt, 38, 40, 43, 141
Oesterle, 82
Okagawa. 141, 142
Opsahl. 131. 149, 151
Orechoff, 29, 145
Orla-Jensen, 64, 141
Oshima, 23, 136
Ostwald. 11, 102, 110, 113, 134
Oudin. 29, 144
Parishev, 29, 147
Pasteur. 9. 10, 11, 12, 13, 19, 20, 21,
25, 35, 59, 73, 133, 134
Pearson, 53, 135
Pederson. 61, 62, 64, 143
Peebles, 137
Pere. 62, 63. 64, 135, 136
Peterson, 61, 62, 143, 145, 147
Pfeiffer, 31, 72, 148
Pictet, 100, 137
Piutti. 134
Podloucky, 151
Pope, 53, 135
Pottevin, 62, 136
Power, 29
Pringsheim, 22, 28, 48, 83, 139, 140,
141
Rabinowitch, 29, 145
Rainey, 15, 148
Rail, 93, 150
Raoult, 11
Raske, 30, 138
Ratner, 61, 153
Rees, G., 150, 151
Rees, W., 112
Rhino, 31, 72, 142
Richter, 146
Ritchie, 37, 50, 53, 145
Rittenberg, 61, 150, 152, 153
Roberts, 83
Rona. 68, 69, 70, 145
Rosenberg, 32, 138
Rosenblueth. 121, 122
Rosenthaler. 38, 70, 139
Roth. 29, 146
Rothen, 73, 148
Rotschy, 100, 137
Ruhland, 24, 25, 144
Sagen, 83
Salkowsky, 23
Samuely, 48, 137
Saneyoshi, 140
Schimmel, 29
Schittenhelm, 48. 138
Schmitz, 71, 140
Schoen, 147
Schoenheimer, 61, 150, 153
Schramm, 153
Schulze, 22, 48, 134, 137
Schwarze, 24, 25, 145
Siegel, 72, 73, 142
Simon. E., 67, 68, 71, 143
Simon, M., 152
Sizer, 115
Smaragdova, 60, 89, 90, 91, 100, 101,
102, 104, 105, 106, 107, 108, 109,
110, 113, 114, 115, 117. 118, 122,
123, 124, 125, 126, 149, 150, 152
Spielmann, 31
Spencer, 136
162
AUTHOR INDEX
Spiers, 53, 149
Stapp, 83
Stiles, 112
Stoklasa, 25, 138
Stoll, 36, 145
Strelitz, 149
Strong, 37, 53, 136
Study, 15, 140
Szent-Gyorgyi, 95
Tatum, 64, 145, 147
Tetzner, 48, 146
Teuffert, 142
Thiele, 25, 140
Thierfelder, 59, 135
Thomas, 120
Tollens, 23, 136
Tomiyasu, 37, 149
Town, 153
Trask, 32
Triem, 43, 46, 47, 53, 147
Tristram, 150
Tromsdorff, 20, 149
Tschugaeff, 32
Turner, 79, 143
Van't Hoff, 134
Vernadsky, 19, 32, 146
Walden, 32, 73, 136, 137
Waldschmidt-Leitz, 129, 132, 151,
153
Walker, 44, 139
Wallach, 29
Warburg, 129
Webster, 147
Weltzien, 27, 141
Wendel, 22, 139
Wetzel, 24, 25, 144
White, 151
Widmann, 71, 144
Wiedemann, 36, 145
Williams, 150, 151
Willstatter, 68
Winterstein, 22, 137
Wohl, 16, 17, 142
Wohlgemuth. 136, 137
Wood, 27, 141
Wyrouboff, 11, 134
Zvetkov, 84, 149
Zycha, 83
Corrigenda
P. 20, 18th line from the bottom, read 1939 instead of 1938.
P. 29, line 13, read Brockmann instead of Blockmann.
P. 59, 8th line from the bottom, read uilxtrorotatoky instead
laevorotatory.
of
Live Music Archive
Librivox Free Audio
Metropolitan Museum
Cleveland Museum of Art
Internet Arcade
Console Living Room
Open Library
American Libraries
TV News
Understanding 9/11